STUDIES ON THE d-GALACTOSl-DASES OF NORMAL AND FABRY PLASMA Thesis for the Degree of Ph.VD. MiCHIGANSTATE UNIVERSITY CAROL A. MAPES 1972 University A _.—.-~—‘4 m 9’ ‘ V BINDING BY HDAE & SUNS' 800K BINDERY INC. uamnv amozns .. - .HG“’.’_WC-‘55Al " :"- - ,,' ABSTRACT STUDIES ON THE a-GALACTOSIDASES OF NORMAL AND FABRY PLASMA By Carol A. Mapes Ceramide trihexosidase activity was discovered in normal human plasma. This enzymatic activity exhibited a bimodal pH optimum, suggesting that there might be two molecular species of ceramide trihexosidase in plasma. This possibility was confirmed when the ceramide trihexo- sidase activity with a pH optimum of 5.4 was separated from the activity with a pH optimum of 7.2 by low temperature ethanol fractionation. The activity with a pH optimum of 5.4 (A form) could be further fractionated from Cohn fraction IV-l and stabilized by a series of steps including ammonium sulfate precipitation of contaminating proteins, treatment with 5% butanol, acetone precipitation, and affinity chromatography. These procedures separated the A form of ceramide trihexo- sidase into two enzymatically active proteins (A-1 and A-Z). The B form of ceramide trihexosidase was purified from Cohn fraction I using the same procedures and was separated into five enzymatically active proteins (8-1, 8—11, B-Ill, B-IV, B-V) by isoelectric focusing. Carol A. Mapes The A—forms of ceramide trihexosidase appeared to be homogeneous as determined by a single band on polyacryl- amide gel electrOphoresis and by the presence of a single protein, coincident with enzymatic activity of constant specific activity, on isoelectric focusing, affinity chromatography, and sucrose density gradient centrifugation. The A forms of the enzyme were activated by sodium taurocholate and sodium chloride. In the absence of these activators the enzymes displayed sigmoidal substrate saturation curves. The sigmoidality was eliminated by the addition of sodium taurocholate and sodium chloride to the incubation mixture. In addition, these enzymes demonstrated the anomalous characteristic of becoming inactive when concentrated. On the basis of inhibitor and activator studies a model for the mechanism of ceramide trihexosidase hydrolysis was presented. This model assumes that the enzyme expresses its Optimum activity when complexed with a mixed substrate-cholate micelle and that excess enzyme causes formation of an inactive enzyme-micelle aggregate. The A-l form of ceramide trihexosidase was competitively inhibited by digalactosylceramide and the trisaccharide obtained by ozonolysis of GL—3. The A-2 form of enzymatic activity was inhibited by the products of the reaction, galactose and lactosylceramide. It was also competitively inhibited by digalactosylceramide, trisaccharide and Carol A. Mapes inositol. Under the conditions of these experiments neither of the enzymes was inhibited by the artificial substrate 4-methylumbellifery1-a-galactoside. The A forms of ceramide trihexosidase have molecular weights of approximately 95,000, are similar in their electrophoretic characteristics, and are specific for the lipid substrate. Neuraminidase treatment of the A-l form of ceramide trihexosidase converted it to an enzymatically active protein which was indistinguishable from the B-V form of ceramide trihexosidase on cellulose acetate electrophoresis. Both [14C]sialic acid and [14C]N-acety1glucosamine could be incorporated into the B-V form of the enzyme, forming several proteins. One of these proteins was electro— phoretically indistinguishable from the A form of ceramide trihexosidase. Thus it was postulated that the two groups of ceramide trihexosidase are glycoproteins related to each other by their sialic acid content. An a—galactosidase affinity column adsorbent was prepared by attaching p-aminOphenylmelibioside to Affinosc 202. This substituted agarose was used to study the enzymes of normal and Fabry plasma. Affinity chromatography of Fabry plasma revealed that the A forms of the enzyme were partially inactive,.whereas all of the B forms of ceramide trihexosidase were absent. In addition, there was an accumulation of catalytically inactive proteins in Fabry Carol A. Mapes plasma. It was suggested that the catalytically inactive proteins were the B forms of ceramide trihexosidase which were converted to the A-l form of the enzyme. Affinity chromatography also revealed that six non- specific a-galactosidases were present in plasma. The specific activities of several of these a-galactosidases were altered in Fabry's disease. Unexpectedly, two of the enzymes hydrolyzing p-nitrophenyl-a-galactoside and 4-methylumbelliferyl-a—galactoside were activated by affinity chromatography. After affinity chromatography these enzymes had 98% of normal activity and could not be used to distinguish hemizygotes, heterozygotes, or normals. A specific enzyme for the hydrolysis of digalactosyl- ceramide was discovered in normal plasma. This enzyme was separated into two active components by isoelectric focusing and cellulose acetate electrOphoresis. In Fabry plasma only the protein of slower electrophoretic mobility was detectable. STUDIES ON THE a-GALACTOSIDASES OF NORMAL AND FABRY PLASMA By Carol A} Mapes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 .u» . v r..\ . .. o . . . . .. b t .w c .¢< ‘ ‘ (9A ACKNOWLEDGMENTS I would like to express my appreciation to Dr. Charles C. Sweeley for his guidance, continual interest and stimulating discussions during the course of my graduate work. I would also like to thank Dr. Clarence H. Suelter for his guidance and helpful discussions concerning the purification and kinetic characterization of the ceramide trihexosidases and to Drs. Richard Anderson, Glen Dawson, Roger Laine and Walter Esselman for their helpful discussions. I would also like to express my appreciation to the following persons: Dr. William Krivit of the University of Minnesota for the use of his laboratory in performing human experimentation and for the many samples of Fabry plasma which he provided; Dr. James Sgouris for providing Cohn fractions; Dr. Graham Jamison for a large-scale preparation of ceramide trihexosidase; Drs. Michel Philippart and Matthew Spence for gifts of Fabry plasma; and Dr. Saul Roseman for providing [14C]sialic acid. Finally, I greatly appreciate the help I have received from the members of the Department of Biochemistry and from Drs. Konrad Sandhoff and Shimon Gatt. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . . . . The Sphingolipid Hydrolases B—Glycosidases B-Galactosidase (Krabbe's Globoid Cell Leukodystr0phy). . . . . B-Galactosidase (Lactosylceramidosis) B-Glucosidase (Gaucher's Disease) Arylsulfatses (Metachromatic Leukodystr0phy). The Hexosaminidases (Tay-Sach's Disease) Purification of B-N-Acetylglucos- aminidases from Beef Spleen . Sialidase Hydrolyzing GMZ Ganglioside a—Galactosidases . . . . Digalactosylceramidase. Ceramide Trihexosidase (Fabry's Disease). . . . . . . . . . . Interaction of Enzymes with Lipid Substrates . . . . . . . . . . . . Multiplicity of Proteins . . . . . . . . . MATERIALS AND METHODS. . . Materials . Sources of Enzymatic Activity. Reagents . . . iii Page 10 10 14 16 20 21 22 22 22 25 29 33 33 33 34 TABLE OF CONTENTS (Continued) Page MethOdS I O O O O O O O O O O O O O O O I O O O O 38 Purification of Lipids . . . . . . . . . . . 38 Isolation of GL-S and GL-Zb . . . . . . 38 Isolation of GL-Za. . . . . . . . . . . 39 Preparation of Phospholipids. . . . . . 39 Chemical Modifications of GL-3 . . . . . . . 40 Preparation of [3H]GL-3 . . . . . . . . 40 Preparation of galactosyl(al+4)- galactosyl(81+4)glucopyranose . . . . . 41 Preparation of GL-Za by Chemical degradation of GL-3 . . . . . . . . . . 41 Enzymatic Syntheses. . . . . . . . . . . . . 42 Preparation of galactosyl(al+6)- galactosyl(a1+6)glucopyranose . . . . . 42 Preparation of Lysolecithin . . . . . . 42 Preparation of [14]GL-3. . . . . . . . . . . 42 Preparation of Trihexosylsphingosine. . 42 Coupling of Trihexosylsphingosine to [14C]Fatty Acids. . . . . . . . . . . . 44 Preparation of d-Galactosidase Affinity Column . . . . . . . . . . . . . . . . . . . 44 Melibiose Octaacetate . . . . . . . . . 44 Acetobromomelibiose . . . . . . . . . . 4S p-Nitr0phenylacetomelibioside . . . . . 45 Deacetylation . . . . . . . . . . . . . 46 Reduction of p-Nitrophenylmelibioside . 46 iv TABLE OF CONTENTS (Continued) Page Coupling Reaction . . . . . . . . . . . 46 Thin-layer Chromatography of Carbohydrate Derivatives. . . . . . . . 47 Identification of Products by GLC . . . 47 Operating Conditions. . . . . . . . . . 48 Preparation of Neuraminidase Affinity Column . . . . . . . . . . . . . . . . . . . 49 Coupling of Tyrosine to Affinose 201. . 49 Reduction of N-(p-Nitrophenyl)oxamic Acid. . . . . . . 50 Coupling Reaction . . . . . . . . . . . 50 Operating Conditions. . . . . . . . . . SO Assays for a-Galactosidase Activity. . . . . 51 Incubation of GL-3 with Crude Enzyme Preparations. . . . . . . . . . . . . . 51 Incubation of GL-3 with Purified Enzyme. . . . . . . . . . . . . . . . . 52 Incubation of GL-3 with Radioactive Substrates. . . . . . . . . . . . . . . 52 Assay in Butanol. . . . . . . . . . . . 53 Incubation of Enzyme with Trisaccharide . . . . . . . . . . . . . 53 Spectrophotometric Quantitation of Liberated Galactose . . . . . . . . . . 54 Quantitation of Liberated Galactose by GLC. . . . . . . . . . . . . . . . . S4 Detection of [3H]Galactose. . . . . . . 56 Detection of [14C]GL—2a . . . . . . . . S6 TABLE OF CONTENTS (Continued) Assays for Digalactosylceramide:Galactosyl Hydrolase. . . . . . . . . . . . . . . Assays for Non-specific a-Galactosidases 4-Methylumbelliferyl-a—galactoside as Substrate. . . . . . . . . . . p-Nitrophenyl-o—galactoside as Substrate . . . . . . . . . . . . . . Plasma Infusions . . . . . . . . Isolation of Ceramide Trihexosidase. Purification of Human Plasma Ceramide Trihexosidase, Form A . . . . . . Pilot-scale Isolation of Ceramide Trihexosidase, Form A . Isolation of Human Plasma Ceramide Trihexosidase, Form B . . . . . . . Isolation of Ceramide Trihexosidase from Human Kidney . . . . . . Miscellaneous Assays . . . . . Protein Determinations. Sialic Acid Determinations. . . . . Molecular Weight Determinations. Sucrose Density Centrifugation. Gel Chromatography. ElectrOphoretic Methods. Isoelectric Focusing. Polyacrylamide Gel Electr0phoresis. vi Page 57 S7 57 58 58 59 59 60 62 62 64 64 64 64 64 65 65 65 66 RESULTS. TABLE OF CONTENTS (Continued) Cellulose Acetate ElectrOphoresis Staining of Activity Staining of Glyc0proteins. Ponceau S Staining for Quantitation of Proteins Interconversion of Ceramide Trihexosidases, Forms A and B. Neuraminidase Treatment of Partially Purified Ceramide Trihexosidases, Form A. . . . . . . . . . . . Treatment of Purified Plasma Ceramide Trihexosidase Form A-l with Neuraminidase Incorporation of £14C]UDP- -N- -Acetyl- glucosamine and [ 4C]CMP Sialic Acid into Ceramide Trihexosidase, Form B- Identification of Radioactive Substrates. Preparation of [3H]GL-3. Preparation of [14C]GL-3 Preparation of a-Galactosidase Affinity Column. Choice of Carbohydrate for Coupling to Affinose 202 . . . . . . . . . Product Identification Specificity for a-Galactosidases Adsorption of Ceramide Trihexosidases. vii Page 66 67 67 68 68 68 69 7O 72 72 72 77 78 78 83 87 87 TABLE OF CONTENTS (Continued) Affinity Chromatography of Neuraminidase. Synthesis of Affinity Column Adsorbent . . Purification of Neuraminidase. . . . . Determination of Products Liberated by Enzymatic Hydrolysis of GL-S. Enzymatic Determination of Liberated Galactose. . . . . . . . . . . . . Determination of Radioactive Hydrolysis Products . . . . . . . . . . . . . . . Occurrence of Ceramide Trihexosidase in Human Plasma. Presence of Ceramide Trihexosidase Activity in Normal Plasma. . . Ceramide Trihexosidase Activity in the Sphingolipidoses . . . . . . . . . . . . . Ileplacement of Deficient Ceramide Trihexosidase IXctivity in Fabry Plasma. . . . . . . . . Substrate and Enzyme Levels in Fabry Plasma Following Plasma Infusion Comparison of Enzymatic Activity Determined by Artificial and Lipid Substrates Following Plasma Infusion. . . . F’urification of Ceramide Trihexosidases Separation of the Plasma Ceramide Trihexosidases into two Enzymatically Active Forms viii Page 89 89 89 94 94 94 96 96 96 100 100 109 112 112 TABLE OF CONTENTS (Continued) Page Isolation of Ceramide Trihexosidase. . . . . 114 Purification Steps . . . . . . . . . . . . . 118 Extraction of Ceramide Trihexosidase, Form A. . . . . . . . . . . . . . . . . 118 Extraction of Ceramide Trihexosidase, Form B. . . . . . . . . . . . . . . . . 118 Extraction of Kidney Ceramide Trihexosidase . . . . . . . . . . . . . 119 Stabilization and Precipitation of the Enzymes . . . . . . . . . . . . . . 119 Affinity Chromatography of Ceramide Trihexosidase, Form A . . . . . . . . . 120 Affinity Chromatography of Ceramide Trihexosidase, Form B . . . . . . . . 123 Affinity Chromatography of Kidney Ceramide Trihexosidase. . . . . . . 128 Other Purification Procedures Evaluated. . . 128 Characterization of the Ceramide Trihexosidases, Form A. . . . . . . . . . . . . . . . . . . . . . 13S Substrate Specificity. . . . . . . . . . . . 135 Investigation of the Enzymes Used in Sequencing GL-3. . . . . . . . . . . . . . . 136 Electrophoretic Properties . . . . . . . . . 140 Estimation of Molecular Weight . . . . . . . 14S Sucrose Density Gradient Centrifugation. . . . . . . . . . . . . 14S Gel Filtration. . . . . . . . . . . . . 145 ix TABLE OF CONTENTS (Continued) Page Heat Stability and Organic Solubility. . . . 148 Effect of Detergents, Salts, and PhOSpho- lipids on Enzymatic Activity . . . . . . . . 153 Kinetics . . . . . . . . . . . . . . . . . . 155 Kinetics of Stimulation of Ceramide Trihexosidase by Sodium Taurocholate and Sodium Chloride . . . . . . . . . . 155 Effect of Enzyme Concentration on the Hydrolysis of GL-3. . . . . . . . . 162 Inhibition Studies. . . . . . . . . . . 165 Classification by Gatt's Kinetic System. . . . . . . . . . . . . . . . . 182 a-Galactosidases of Whole Plasma. . . . . . . . . 186 Affinity Chromatography of Normal Plasma . . 186 Affinity Chromatography of Fabry Plasma. . . 189 Investigation of Digalactosylceramide: Galactosyl Hydrolase in Human Plasma . . . . 197 ElectrOphoretic Investigation of Digalactosylceramide:Galactosyl Hydrolase . . . . . . . . . . . . . . . 197 Urinary Ceramide Trihexosidases . . . . . . . . . 204 Interconversion of the Ceramide Trihexosidases. . 204 Preliminary Neuraminidase Experiments. . . . 204 Neuraminidase Treatment of Purified Ceramide Trihexosidase, Form A-l . . . . . . 209 DISCUSSION SUMMARY. BIBLIOGRAP APPENDIX TABLE OF CONTENTS (Continued) Preliminary Studies on Incorporation of Sialic Acid into Ceramide Trihexosidase, Form B-V Incorporation of El 4C]UDP- -N- -Acety1- glucosamine and [ 4C]CMP-Sia1ic Acid into Ceramide Trihexosidase, Form B- V. HY . . . . xi Page 221 221 230 256 258 271 TABLE 10 ll .12 125 1<1 LIST OF TABLES Page Nomenclature of the Sphingolipids and Sphingolipid Hydrolases .‘. . . . . . . . . . 8 Enzymes Having Multiple Forms in Human Blood . . . . . . . . . . . . . . . . . . . . 31 Rf of Carbohydrate Derivatives. . . . . . . . 86 Specificity of a-Galactosidase Affinity Column. . . . . . . . . . . . . . . . . . . . 88 Purification of Clostridium perfringens Neuraminidase . . . . . . . . . . . . . . . . 95 Ceramide Trihexosidase Activity in Human Plasma. O O I O O I O O O O O O O O O O O O O 99 Concentration of Glycolipids in Fabry Plasma Following Infusion of Normal Plasma. . 105 Ceramide Trihexosidase Activity in Cohn Fractions . . . . . . . . . . . . . . . . . . 113 Purification of Human Plasma Ceramide Trihexosidase, Form A . . . . . . . . . . . . 115 Purification of Human Plasma Ceramide Trihexosidase, Form B . . . . . . . . . . . . 116 Purification of Human Kidney Ceramide Trihexosidase, Form A . . . . . . . . . . . . 117 Comparison of Enzymatic Activity using Natural and Artificial Substrates . . . . . . 137 Effect of Detergents on the Specific Activity of Ceramide Trihexosidase. . . . . . 154 Effect of Salts on the Specific Activity of the Ceramide Trihexosidases. . . . . . . . 156 xii TABLE 15 16 17 18 LIST OF TABLES (Continued) Effect of PhOSpholipids on the Specific Activity of the Ceramide Trihexosidases. Summary of Kinetic Parameters. Summary of the Protein and Activity Obtained for the Individual a- Galactosidases Following Affinity Chromatography . . . Comparison of the Plasma a-Galactosidase Activity Before and After Affinity Chromatography . . . . . . xiii Page 157 183 193 196 FIGURE 111 122 113 141 1~53 LIST OF FIGURES The Current Concept of Glycosphingolipid Metabolism. . Possible Kinetic Curves for Enzyme Inter- actions with Lipid Substrates Strip Scan of [3H]GL-3. GLC of TMSi Methyl Glycosides from [3H]GL-3 Strip Scan of [14C]GL-3 Enzymatic Hydrolysis of Oligosaccharide Substrates. . . a-Galactosidase Affinity Column Adsorbent Affinity Chromatography of the A forms of Ceramide Trihexosidase on Unsubstituted Affinose 202. Neuraminidase Affinity Column Adsorbent Effect of pH on Ceramide Trihexosidase Activity in Normal Plasma . Levels of Enzymatic Activity and Substrate Concentration in Fabry Plasma Following Infusion of Whole Plasma. Comparison of Enzymatic Activity at pH 5.4 and pH 7. 2 in Fabry Plasma Following Infusion of Normal Plasma . . Plasma a-Galactosidase Activity in a Patient with Fabry's Disease after Infusion of Normal Plasma . Affinity Chromatography of Plasma Ceramide Trihexosidase, Form A . . . . . . . . . Affinity Chromatography of Plasma Ceramide Trihexosidase, Form B . . . . . . . . xiv Page 27 74 76 80 82 85 91 93 98 104 107 111 122 125 FIGURE 16 17 18 19 20 21 22 23 241 255 26 (A) 26 (B) 217 28 29 (A) LIST OF FIGURES (Continued) Cellulose Acetate Electrophoresis of Plasma Ceramide Trihexosidase, Form B. Isoelectric Focusing of Plasma Ceramide Trihexosidases, Form B. Affinity Chromatography of Human Kidney Ceramide Trihexosidase. . . Comparison of Kidney and Plasma Ceramide Trihexosidases, Form A, by Cellulose Acetate Electrophoresis . . . Substrate Specificity of Plasma Ceramide Trihexosidases, Form A. Affinity Chromatography of Ficin. Isoelectric Focusing of the Plasma Ceramide Trihexosidases, Form A. Scans from Polyacrylamide Gel Electrophoresis of Ceramide Trihexosidase A-1 and A-2 Molecular Weight Determination by Gel Filtration. . . . . . . . . . . . Heat Stability of the Ceramide Trihexosidases, Form A-l (A) and Form A-2 (B) Lineweaver-Burk Plot Showing the Effect of Sodium Taurocholate on Ceramide Trihexosidase, Form A-l. Lineweaver-Burk Plot Showing the Effect of Sodium Taurocholate on Ceramide Trihexosidase, Form A-Z. Effect of Enzyme Concentration on the Hydroly- sis of GL- 3 . . . . . . . . . . . The Effect of GL- 2b on the Hydrolysis of GL- 3. . . . Lineweaver-Burk Plot Showing the Inhibitory Effect of Trisaccharide on Ceramide Trihexosidase, Form A-l (C?) Page 127 130 132 134 139 142 144 147 150 152 159 161 164 167 169 FIGURE 29(3) 30(A) 30(B) 30(C) 30(D) 30(5) 3]. 322 313 311 343 15(3 3V7 LIST OF FIGURES (Continued) Lineweaver-Burk Plot Showing the Inhibitory Effect of GL-Zb on Ceramide Trihexosidase, Form A-l. Lineweaver-Burk Plot Showing the Inhibitory Effect of GL—2a on Ceramide Trihexosidase, Form A-Z. Lineweaver-Burk Plot Showing the Inhibitory Effect of Galactose on Ceramide Trihexosidase, Form A-2 Lineweaver-Burk Plot Showing the Inhibitory Effect of Trisaccharide on Ceramide Trihexosidase, Form A-Z Lineweaver-Burk Plot Showing the Inhibitory Effect of GL-Zb on Ceramide Trihexosidase, Form A-Z. Lineweaver- Burk Plot Showing the Inhibitory Effect ofm myo -Inositol on Ceramide Trihexosidase, Form A- 2 . The Effect of Increasing Trisaccharide Concentration on Ceramide Trihexosidase Activity. Affinity Chromatography of Whole Plasma Comparison of Normal and Fabry Ceramide Trihexosidase by Cellulose Acetate Electrophoresis Effect of pH on the Hydrolysis of GL-Zb Affinity Chromatography of Digalactosyl— ceramide: Galactosyl Hydrolase . Isoelectric Focusing of Digalactosylceramide: Galactosyl Hydrolase. Comparison of Normal and Fabry Digalactosyl— ceramide:Ga1actosyl Hydrolase Activity by Cellulose Acetate Electrophoresis xvi Page 171 173 175 177 179 181 185 188 190 199 201 203 206 FIGURE 38 39 40(A) 40(3) 41 42 43 44 45 LIST OF FIGURES (Continued) Affinity Chromatography of Concentrated Urine . . . . . . . . . . . . . . . . . Effect of Neuraminidase on the pH Optimum of the Ceramide Trihexosidases, Form A . Correlation of Sialic Acid Release with Changes in Enzymatic Activity Correlation of Sialic Acid Release with Changes in Enzymatic Activity . Cellulose Acetate Electrophoresis of Ceramide Trihexosidase, Form A-l after Neuraminidase Treatment Isoelectric Focusing of Ceramide Trihexo- sidase, Form A-l, after Neuraminidase Treatment Cellulose Acetate Electrophoresis of Ceramide Trihexosidase, Form B-V, following Sialyl- transferase Treatment . . . . . . Incorporation of [14C]Sialic Acid into Ceramide Trihexosidase, Form B-V. Incorporation of [14C]UDP-N-Acetylglucosamine into Ceramide Trihexosidase, Form B-V . . xvii Page 208 211 213 215 218 220 224 226 229 INTRODUCTION Thudichum published the first report on the occurrence of Sphingolipids in brain in 1874 (1). Approximately 15 years later he summarized his life-work on the discovery and primary chemical characterization of these lipids which included cerebrosides, sphingomyelin, and sphingosine (2). The second phase of Sphingolipid work occurred during the first four decades of this century. These studies, carried out primarily by Thierfelder, Klenk, Levene and Rosenheim (3), were concerned mainly with the isolation and detailed structural characterization of the individual lipids. During this period the sulfatides were discovered by Blix (4) and a class of complex acidic glycosphingolipids, gangliosides, was reported by Klenk (5-7). Experiments on the degradation of Sphingolipids by tissue preparations were first reported by Thannhauser and Reichel (8,9). These as well as other investigators used Crude preparations to degrade cerebrosides (8,10), SPhingomyelin (9,11,12), sulfatides (13), and gangliosides (14). The first experiments with partially purified enzymes fronlnmnmmlian tissues appeared in the 1960's and described the hydrolysis of ceramide from brain and of sphingomyelin by enzymes from brain and liver (16,17). These studies were followed by reports on the isolation and properties of enzymes which catalyze the hydrolysis of ceramide (18,19), galactosylceramide (27-29), several glycosphingolipids having a terminal galactose residue (24, 30-32), N-acetyl- galactosamine (33, 34) and N—acetylneuraminic acid (35-37). The cumulative results of these and other studies, as illustrated in Figure l, were organized into a pattern consisting of a multi-step successive degradation of mono- saccharide units which could account for the catabolism of glycosphingolipids to ceramide (N-acylsphingosine). The hydrolysis of ceramide to sphingosine and fatty acids, catalyzed by a ceramidase isolated from rat brain (38), results in the formation of Sphingolipid bases which can be metabolized to either palmitic acid or pentadecanoic acid, both of which can be degraded by the B-oxidation and tricarboxylic acid systems. Thus a combination of these pathways may account for the total degradation and oxidation 0f most mammalian glycosphingolipids. During the same periods of time when these lipids were first being discovered and characterized, reports of rare diseases, now know to be Sphingolipidoses, were appearing hi the literature. In the late 1800's Anderson (39) and Fabry (40) independently described the clinical E5Y1n‘ptoms of patients with angiokeratoma corporis diffusum (Fabry's disease). Reports on other patients having similar .mZOAAm :oxoun >2 woumofimcfi ohm .momowfimwfiomcflgmm ecu mo oco ca mcfiuasmop .oEcho owaonmumo m mo xocoflofiwov m m« when“ gofinz um mopflm .mommwflmooxam mo :ofiuom ecu xn xfio>fimmooo3m wovmhmow ohm mvfimfiaowcflgmmooxaw KonEoo ozk Emflaonmuoz wflmflfiowcfigmmooxfiu mo umoocoo Osmansu osb .H oADMHm «0.5030 848:. :H: 95.12.40 4. so: .8: 80:0.0 50:.00 .8: .8485 50.5.34... 4. 4 mm: $405909.an 35.5.2384 4.8: o : ..: 0:428:04»; .8 so: .8 .8: .8 .8 8.1.8.8 4242: 842.8: .8: 848.0 9554... .00:o.0:.00: 8.48.0 6 808.2» .00: :20: :.00:uA< ommvwmopomflmu-m owfimonoau owfiEMHoonH+Hmuaxmoo3Hw -m4+Hm.meouomHmm -54+H0.H>mouomammmm+amv -HxaflsmmouomHmMquoom:z N20 onfim< 00wEmhoofiH+Hmvaxmoosfim -.¢+Hm.fixmooomfiam.e+flm. -H>:HEmmouomHmmH%uoom-z N20 owfiemhooma+amvaxmoosam -.4+Hm.fismoouafiam..m+m. -HKCMEmHDoCquoom-z.n4+amv -chfismmOpomHmwaxuoom-z m-qu o©HEmaoomH+Hmvaxmoosam -fl4+Hm.meouomHmm -A4+H0.meouomfimo EN-40 owfiEmhoomH+HmvaxmouomHmm -fl4+Hc.meouomHmo mm-40 owflEmpoonH+HmvfixmoUSHm -.¢+Hm.fixmooumaao meauamfism meaeaaooflfi+flm. -HomHSm-m.H>mouomHmw £0-08 measanouflfi+H80HAmopumHau m.:omm-me m.»pnmm mflmo0HEmpo0 :meouomq >nmouumxmoxsoq ufiumEounomuoz m.onnmhm ommwfim000H0-m «H-40 ovflEmhoomH+Hmvaxmoosau m.noaozmu oexncm cofiumfi>onnn< Uwaommumu 0mm“; mmvvfimfiq woumasesoo< ommomwn mommaouwxm wanfiaowzflgmm 000 mvfimfifiownfinmm ozu mo ouduwaucosoz .H oHan w— . ~ _ _ summarizes the nomenclature of the substrates and enzymes for each of the sphingolipidoses to facilitate the discussion of the individual enzymes. B-GLYCOSIDASES B-Galactosidase (Krabbe's Globoid Cell Leukodystr0phy): Austin et al. reported that the activity of cerebroside- sulfatide sulfotransferase was deficient in the gray and white matter of brain and in the kidney of two patients with globoid cell leukodystrOphy when compared to normal controls (60, 61). Cerebroside-sulfatide sulfotransferase is the enzyme which catalyzes the formation of sulfatide from cerebroside and active sulfate, phosphoadenosine phosphosulfate (62-64). Other laboratories originally were unable to confirm this finding (65, 66), but it now appears that cerebroside- sulfatide sulfotransferase is moderately depressed in globoid cell leukodystrophy although it is not the primary genetic defect (67). A deficiency of galactocerebroside-B-galactosidase activity (pH optimum 4.5) has been demonstrated in the gray and white matter of brain, liver, kidney and spleen of Krabbe patients (67-69). Activity is also deficient in the peripheral leukocytes (70, 71), serum (70) and cultured fibroblasts (70) when measured with the natural lipid substrate [3H]galactosylceramide. 10 It appeared that dogs of the Cairn and West Highland terrier family might serve as models for this disease, but their enzymes, at least in the serum, appear to be completely different from those of the human (72). In both the human and canine globoid cell leukodystrophy, there is no difference in the enzymatic activity of normals, heterozygotes, or affecteds when measured with the artificial substrate, 4-methy1- umbelliferyl-B-galactoside (72), thereby indicating that the enzyme is specific for the lipid substrate. The instability of this enzyme has prevented any further studies on its properties and has defeated all attempted isolations. B-Galactosidase (Lactosylceramidosis): Dawson and Stein recently detected a previously unreported sphingolipidosis characterized by an elevation of GL-Za in erythrocytes, plasma, bone marrow, urinary sediment, liver biopsy, and brain biopsy (73). This paper also reported that lactosyl- ceramide:galactosyl hydrolase activity (pH optimum 5.0) in the liver of this patient, measured with [3H]GL-2a, was 15% of that of normal liver. B-Glucosidase (Gaucher's Disease): In adult Gaucher's disease there is a deficiency of B—glucosidase activity which hydrolyzes GL-la (glucocerebroside) in leukocytes (74,75), cultured skin fibroblasts (76,77), liver and spleen (78,79). These deficiencies have been detected using both natural and artificial substrates although there is some discrepancy in the results obtained with these substrates. 11 In the spleen the defect has been demonstrated by using glucocerebroside and p-nitrophenyl-B-glucoside as substrates (80,81). In the liver however, the two activities do not parallel each other since the p-nitrophenyl-B-gluco- sidase activity is increased while that of glucocerebrosidase is decreased (81). In contrast to these findings it was reported that liver homogenate from a patient with Gaucher's disease was inactive in the hydrolysis of either 4-methyl- umbelliferyl-B-glucoside or GL-la (82). Recent studies comparing the reactivity of B-glucosidase with natural and artificial substrates have proved that, at least in leukocytes, the enzyme specifically hydrolyzes only GL-la (83). This conclusion was based on the results of leukocyte assays in two different patients. In one case an individual diagnosed as a Gaucher on the basis of histo- chemical studies was found to have normal levels of enzymatic activity when measured with the artificial substrate, but incubation of the leukocytes with [14C]g1ucocerebroside revealed depressed B-glucosidase activity. In the second case, diagnosed as a Gaucher on the basis of decreased activity toward 4-methylumbelliferyl-B-glucoside, the patient was normal when diagnosed with radioactive lipid substrate. Kanfer and associates (83) summarized their conclusions on artificial substrate assays as follows: 12 "The assay of B-glucosidase either as the 4-methy1umbe11iferyl- or p-nitrophenyl- glucoside derivative usually presents difficulties. All determinations were routinely carried out in triplicate in this laboratory. The problem has been that in 5 consecutive attempts to assay a sample, 300% variation can occur in the triplicates. The sixth attempt may be satisfactory ....... ... It should be mentioned that one accurate diagnosis has been obtained with 4-methy1- umbelliferyl-B-glucoside." Studies were recently reported by H0 et a1. (78) on some properties of normal spleen B-glucosidase and reconstitution of B-glucosidase activity with two factors, one derived from normal spleen and one from Gaucher spleen. Normal spleen B-glucosidase activity, measured with 4-methylumbellifery1-B-g1ucoside at pH 4.0 to 4.3, was stimulated about 40% by the addition of 0.02% Triton X-100. Gel filtration on Sephadex G—50 separated the B-glucosidase activity into two proteins. One of these proteins, active at pH 4.0, showed 100% stimulation with Triton X-100, whereas the second protein, enzymatically active between pH 5.0 and 7.0, showed no appreciable detergent activation. The B-glucosidases prepared in an identical manner from Gaucher spleen were also separated into acidic and neutral proteins which differed from the normal enzymes in both pH optima and a lack of stimulation by Triton X-100. The profile of enzymatic activity from the Sephadex column appeared to separate the acidic protein into two enzymes both having negligible activity toward 4-methylumbelliferyl-B-g1ucoside, 13 whereas the neutral glycosidase, reportedly uneffected in Gaucher's disease, appeared to have approximately 50% normal activity. Neither the quantity of protein nor the weight of the spleens from which these proteins were prepared was reported, so the apparent deviations from normal values may represent differences in protein quantity rather than in enzymatic activity. When homogenates prepared from normal and Gaucher spleens were mixed there was an enhancement of B-glucosidase activity 2-3 times greater than expected from theoretical calculations. This "reconstitution" of B-glucosidase activity was attributed to two factors referred to as P, an acid glycoprotein derived from Gaucher spleen, and C, a low molecular weight, particulate, thermolabile substance derived from normal spleen. "Purified" factor P (Gaucher), having no enzymatic activity, was mixed with a crude preparation of factor C (normal) which had negligible B-glucosidase activity. Following the incubation there was reconstitution of B- glucosidase activity. This phenomenon did not occur when factor P (Gaucher) was incubated with factor C (Gaucher) that was prepared in the same way as factor C from normal spleen. During the incubation the disappearance of factor P (factors P and C are separated by centrifugation; factor C sediments) indicated that it was incorporated in some form into the defective Gaucher B-glucosidase causing 14 reconstitution of normal enzymatic activity. It was concluded that Gaucher's disease may involve the deficiency of factor C which is involved in the formation of acid B-glucosidase in association with another protein, factor P. The presence of factor P in normal spleen may indicate that a reaction between the two factors occurs under normal physiological conditions. The reconstituted B-glucosidase hydrolyzed artificial substrates, but its activity against glucocerebroside has not been tested. ARYLSULFATASES (METACHROMATIC LEUKODYSTROPHY) Three forms of arylsulfatase, designated as A, B and C, have been reported (84). Only arylsulfatase A has been purified in substantial amounts, and it is the only one for which at least one natural substrate (cerebroside sulfate; sulfatide) has been identified (85). Arylsulfatase A in combination with a heat-stable complementary factor cleaves sulfate from sulfatide (86,87). Arylsulfatase A has been partially purified from ox liver (88), ox brain (89), pig kidney (90), human brain (91,92), and human liver (93). Recently Breslow and Sloan purified arylsulfatase A l75-fold from human urine using an affinity column adsorbent with a substrate analogue, Psychosine sulfate, as the ligand (94). This protein, which aPpeared as a single band on polyacrylamide electrophoresis, was assumed to have a monomeric molecular weight of 110,000 fron1gel filtration studies on Sephadex G-200. The amino Y . 3|) t ) .- b-ol n-. v... s ‘fi ‘5 ll) ‘7- s.‘\ 1’) _I 'c 4 ~ 15 acid composition of the protein was reported with a large portion of the residues being glutamic acid, proline and glycine, while methionine was the only sulfur-containing amino acid present. There was no indication by these authors that arylsulfatase A is a glycoprotein, although earlier work by Goldstone at al. showed that neuraminidase treatment of arylsulfatase A resulted in a protein of reduced electro- phoretic mobility which reacted as arylsulfatase B on bio— chemical analysis (95). Antibody to human arylsulfatase A was prepared by injecting highly purified enzyme into rabbits, then rendering it monospecific by absorption with liver fractions which were isoelectrically focused next to the enzyme (93). This anti— body increased the rate at which the normal enzyme hydrolyzed the artificial substrate (p-nitrocatechol sulfate) and stabilized it against heat inactivation. Inactive human liver arylsulfatase A, indistinguishable from the normal enzyme by immunodiffusion and immunoelectro- phoresis, combined with this antibody to form an enzymatically active protein (93). A similarly prepared goat antibody to human arylsulfatase A was found to inhibit enzymatic activity at high concentrations but not at low concentrations (96). Many of the classical antibody experiments were Perfbrmed on arylsulfatase A derived from ox (97) but this enzyme is substantially different from that of the human in 16 electrophoretic characteristics (93,98) and inhibition (99). Goat and rabbit antibodies to the human arylsulfatase A cross-react with monkey and dog but not with sheep, ox or mouse enzymes (96). The possibility was raised that the arylsulfatase A deficiency in metachromatic leukodystrophy might be caused by a deficiency of a sialyltransferase (95). This was based on studies with rat sulfatases which suggested that sulfatase B might be sulfatase A minus sialic acid. However there is no cross-reaction between human sulfatases A and B (96). The question of whether sialic acid residues would account for this immunological difference has not been investigated. THE HEXOSAMINIDASES (TAY-SACH'S DISEASE) Tay-Sach's disease shows an accumulation of GMZ ganglioside (100,101) and its sialic acid-free component, asialo GMZ ganglioside (102,103), also commonly referred to as trihexosylceramide. In most sphingolipidoses the accumulation of a specific sphingolipid is caused by a deficiency of an enzyme for catabolism of the accumulated lipid. By analogy, Tay-Sach's disease should be caused by a deficiency of a catabolic enzyme. Early studies with eXtracts from calf brain proved that N-acetyl-B- hexosaminidase, possessing both N-acetyl-B-glucosaminidase and N-acetyl-B-galactosaminidase activity, hydrolyzed these gangliosides (34). Thus it might be assumed that l7 hexosaminidase is altered in cases of Tay-Sach's disease, however a complete deficiency of this enzyme is found in only one type of the disease which is characterized by the visceral storage of an additional lipid, globoside (104). Hexosaminidase activity consists of two major components designated A and B (105) having isoelectric points of 5.0 and 7.3, respectively (106). Both enzymes A and B degrade asialo GMZ ganglioside and globoside whereas only component A degrades the main storage compound, GMZ ganglioside (107). Investigation of the enzymatic activity in autopsied brain tissue led to the recent discovery of three variants of Tay-Sach's disease (108). Variant O is characterized by an absence of both hexosaminidases A and B (108). Variant B (classical Tay-Sach's disease) is characterized by an absence of hexosaminidase A (109,110). Variant AB is characterized by an enhancement of both hexosaminidases A and B in brain extracts (108). Several investigators have demonstrated a deficiency of hexosaminidase A by starch gel electrOphoresis (109), iso- electric focusing (108,110) and cellulose acetate electro- phoresis (11). In addition the absence of enzymatic activity for hydrolysis of the N-acetylgalactosyaminyl moiety was demonstrated through the use of specifically labeled GMZ ganglioside (112). Hexosaminidase A activity was found to be completely deficient in cultured skin fibroblasts, Peripheral leukocytes and serum of patients with Tay-Sach's 18 disease, as measured for N-acetyl-B-glucosaminidase activity (111). O'Brien et al. reported similar results using an enzyme assay based on the thermal lability of hexosaminidase A (113). Sandhoff at al. (114) have reported the following characteristics for the hexosaminidases: 1) both hexos- F_1 aminidases A and B have a similar pH dependency with optimal activity around pH 4.5; 2) both enzymes have a molecular weight of 130,000 as determined by gel filtration on i Sephadex G-150; 3) the B form is stable for 30 minutes at 50° C in l M acetate buffer, pH 5.0, whereas the A form loses 60% of its activity under these conditions; 4) sodium chloride and crude sodium taurocholate activate the enzymes, while purified sodium taurocholate, Triton X-100 and Cutscum are not stimulatory; 5) the Km's for hydrolysis of p-nitro- phenyl-N-acetyl-B-glucosamine, p-nitrophenyl-N-acetyl-B- galactosamine and asialo GM2 ganglioside are 0.67 mM, 0.16 mM and 0.2 mM, respectively. These data were collected using 4000-fold purified hexosaminidase A and 2000-fold purified hexoaminidase B. In contrast to these results, Brady reported that the Km for the hexosaminidases, purified 6000-fold, was one order 0f magnitude higher for asialo GMZ ganglioside than for the artificial substrates (115). Although hexosaminidase activity consists of two primary Components, Sandhoff reported that both impure and purified 19 preparations of N-acetylhexosaminidase from pig kidney, calf brain, rat brain, and human brain could be resolved into at least four components by isoelectric focusing (106). This observation was supported by Young et al., who resolved human brain hexosaminidase into multiple forms by chromatography on DEAE-cellulose (116). There is evidence that hexosaminidase A from human spleen (105) and human kidney (117) is an acidic glycoprotein which owes much of its charge to sialic acid residues. The effect of neuraminidase on "purified" hexosaminidase A was to produce a number of forms, each showing a stepwise decrease in anodic mobility. If sufficient neuraminidase was added the conversion of Form A into Form B took place without the intermediate stages being detectable. The change in electrophoretic mobility was roughly the same at each successive step, and on this basis it would require at least 12 molecules of sialic acid to be removed to explain the transformation of hexosaminidase A into the basic form B (105). Similar results have been reported by Goldstone et al. (95). Since no evidence has been found to indicate any significant difference in molecular weight of Forms A and B, Form A could be envisaged as a glycoprotein consisting of Form B with up to 12 short carbohydrate side chains, each terminating with a sialic acid residue. 20 Purification of B-N-Acetylglucosaminidases from Beef Spleen: Two enzymes designated A and B, each with N-acetyl- glucosaminidase and N-acetylgalactosaminidase activity, were purified from beef spleen extracts (118). Homogeneity of the enzymes was confirmed by gel electrophoresis and ultra- centrifugation. The authors reported these enzymes to have the following characteristics: 1) only enzyme A was found by DEAE- cellulose at pH 7.0; 2) the enzymes had identical weight- average and z-average molecular weights; 3) the z-average molecular weight was unchanged by addition of guanidine hydrochloride or dithiothreitol, but decreased when both were present, indicating more than one peptide chain in each enzyme; 4) amino acid compositions were similar, but more sialic acid and neutral carbohydrates were present in A than in B; 5) Km values were identical but Vmax was slightly greater for Form A; 6) changes in pH affected the Km and Vmax of both enzymes; 7) the two p-nitrophenyl substrates competed for the active sites on both enzymes; 8) stimulation by bovine serum albumin enhanced vmax but did not affect Km; 9) both enzymes were inhibited by Ag+, “8+2, Fe+2 3 and Fe+ in a noncompetitive manner; 10) incubation of the enzymes With low concentrations of dithiothreitol reduced enzymatic activity; 11) N-ethylmaleimide, iodoacetamide and iodoacetate did not affect activity but p-chloromercuribenzoate was an inhibitor; and 12) EDTA did not cause a significant change if! activity. 21 Sialidase Hydrolyzing GMZ Ganglioside: A particulate enzyme that catalyzes the hydrolysis of N-acetylneuraminic acid from GM2 ganglioside has been found in various tissues of the rat (119). Preparations of enzyme from the small intestine were used to delineate the following properties of this enzyme: 1) Optimal enzymatic activity occurs at pH 5.0; 2) enzymatic activity is inhibited by sodium tauro- cholate, Triton X-100, p-chloromercuribenzenesulfonate and 2 a+2 +2 3 cations including 2n+ , c , Cu , and Fe+ ; 3) the Km for GMZ ganglioside was 0.53 x 10'4 M; and 5) the time course for the sialidase reaction proceeded with a lag phase which could not be removed by prior incubation of the enzyme in the absence of substrate or explained by a two-step sequence in which the N-acetylgalactosaminyl residue was cleaved prior to release of sialic acid. Previously described ganglioside sialidases obtained from rat liver (37) and brain (36,120) were ineffective in catalyzing the hydrolysis of GMZ ganglioside. It now appears that enzymatic hydrolysis of this lipid could theoretically proceed via the removal of the N-acetylgalactosaminyl moiety or, alternatively, by the sialidase reaction. The lipid product of the alternative reaction would be GMS ganglioside. Frohwein and Gatt (34) described an enzyme in calf brain Which catalyzes this reaction. The physiological significance of the two alternative Pathways, the N-acetylhexosaminidase and sialidase reaction, fol? catabolism of GM2 ganglioside in Tay-Sach's disease have 22 been discussed by Brady (119) but there is not evidence that GM3 ganglioside accumulates in this sphingolipidosis. a-GALACTOSIDASES Digalactosylceramidase: Although digalactosylceramide (CL-2b) accumulates in Fabry's disease (49, 50) there has been no evidence that there is an existing a-galactosidase specific for its hydrolysis. There has also been no evidence that ceramide trihexosidase hydrolyzes GL—Zb. Ceramide Trihexosidase (Fabry's Disease): Brady et a2. discovered ceramide trihexosidase in normal small intestinal mucosa and demonstrated its absence in tissue from Fabry patients (51), thereby showing that the accumulation of GL—3 in Fabry's disease is due to an enzyme deficiency. This work is supported by several recent reports demonstrating an absence of ceramide trihexosidase activity in Fabry leukocytes (52-54), cultured skin fibroblasts (55-57), amniotic fluid (55), kidney (32, 58), spleen (32), brain (32), and liver (32). Relatively few reports concerning the characteristics of ceramide trihexosidase have been published. The enzyme, originally isolated from rat intestinal tissue by Brady et aZ., occurred as a single protein having optimal activity at pH 5.0, as measured with [3H]GL-3 (32). The addition of SOdium cholate (2 mg/ml) enhanced enzymatic activity which Occurred with a linear rate of hydrolysis. Half-maximal 23 velocity for the rat ceramide trihexosidase was 3.7 x 10'4 M. The same assay conditions were used by Brady to detect human ceramide trihexosidase activity (51). The rate of hydrolysis was nonlinear with the human enzyme but the addition of beef spleen extract (unknown composition) corrected the non-linearity. More recent investigations, using 4-methylumbelliferyl- a-galactoside as substrate, have demonstrated that ceramide trihexosidase consists of two components (121,122). Beutler and Kuhl studied the a-galactosidase activity of fibroblasts and leukocytes from both normal and Fabry patients and found the normal a-galactosidase activity to be composed of two isozymes. A thermolabile, low Km component, electro- phoretically rapid at pH 7.0, was designated as a- galactosidase A and a second, smaller fraction designated a-galactosidase B, had a higher Km, greater thermal stability and slower electrophoretic mobility. In Fabry's disease, only the B isozyme could be detected and it was indistinguishable from the normal B isozyme (121). Crawhall and Banfalvi, also using 4-methy1umbelliferyl- a-galactoside as substrate, concluded that in normal cultured skin fibroblasts there are two a-galactosidases (122). This conclusion was based on the fact that myo-inositol appeared to be a competitive inhibitor of normal but not of the residual activity (ls-20% normal) in fibroblasts obtained from patients with Fabry's disease, thereby suggesting that _, -JJ :.‘. .- ‘w ‘1 ‘ ~: ‘1 24 normal fibroblasts contain two a-galactosidases, only one of which is present in cells from patients with Fabry's disease. The a-galactosidase activity in normal cell lines was rapidly inactivated at 51° over a period of 60 minutes. After longer periods of heating, the rate of heat inactivation was much slower and closely paralleled that found for the residual enzyme activity in the cell strains from patients with Fabry's disease. Myo-inositol appeared to be a competitive inhibitor of the normal enzyme, whereas enzymatic activity present in cell extracts from patients with Fabry's disease was not inhibited by myo-inositol, but was mildly stimulated by its addition. The residual activity in Fabry cells had a Km of l4-29 mM, while the Km for normal enzymatic activity was 3-4 mM. A third report on a-galactosidase activity purified SOO-fold from placenta concerned the kinetic preperties and enzymatic alterations in Fabry's disease (123). The following kinetic properties were reported: 1) hydrolysis of [3H]GL-3 exhibited a sigmoidal substrate saturation curve and was competitively inhibited by lactosylceramide (GL-Za); 2) a mixed type of inhibition was observed in the presence of the synthetic substrate, 4-methylumbe11iferyl-a-galactoside; and 3) digalactosylceramide (GL-Zb) was stimulatory at low substrate concentrations and inhibitory at high substrate concentrations. To explain these observations, the author proposed that this a-galactosidase is an enzyme with an effector site 93) f I .4. w". 25 accessible to glycolipids only and a catalytic site accessible to a wide variety of galactosides. INTERACTION OF ENZYMES WITH LIPID SUBSTRATES Information on the mechanism of action of the sphingo- lipid hydrolases is negligible. Most kinetic studies have been limited to determinations of Km and Vmax- Gatt has investigated the interaction of lipid enzymes with pure substrates and classified these interactions into four main types (124). Figure 2 shows the typical kinetic curves obtained for each of the four classifications. Type I has a classical Michaelis-Menton type V/S curve. It is obtained in cases of enzymes acting on lipid substrates where, under the experimental conditions used, there is no monomer-micelle transition. This type of interaction occurs with many enzymes acting on lipid substrates solubilized in detergents. This type of curve may also be obtained when the critical micellar concentration (CMC) of the substrate is low enough that the assay procedure does not detect deviations from the hyperbolic shape below the CMC. Type II has a V/S curve which is hyperbolic to a certain substrate concentration, then breaks and becomes parallel to the abscissa. Similarly, a presentation of the data in the fOrm of a double reciprocal plot has a straight line parallel to the abscissa and a second straight line with a upward Slope. This is the case where the enzyme utilizes monomeric bUt not micellar forms of the substrate. The curve becomes 26 .uXou exp :0 wommsomflw mew 00m Ava. x003 m.pumu Eoem kfiuoopflw coxmp who; mowsmflm omozH moumhumnsm wfimflq cpflz mcofluompoucH oEchm pow mo>hsu OHHOCHM ofipammom .N oeswflm 27 a at E ea... 2 on? H 2...... m§> 28 parallel since the concentration of monomer above the CMC is constant. This monomeric concentration remains constant and equals the CMC even when substrate molecules are removed by the action of the enzyme due to a rapid disaggregation of the micelles to maintain an equilibrium between monomeric and micellar forms. If the "activity" of one micelle equals that of a monomer, this type of interaction would be indistinguishable from Type I. Type III has a V/S curve which is hyperbolic to a certain concentration, then breaks and shows a decreased rate of hydrolysis. In the double reciprocal plot the straight line breaks and is succeeded by a second straight line with an upward slope. This is the case where the enzyme utilizes monomers but probably not micelles. This type occurs when the micelles inhibit the enzyme or interfere with the action of the enzyme on the substrate monomer. Type IV has a V/S curve which is sigmoidal although the sigmoid is usually not symmetrical. This is the case where the enzyme utilizes micelles but not monomer. When considering these classifications two factors ShOUId.be considered: 1) Below the CMC, values on the absCissa are correctly presented, since the concentration 0f the substrate available for interaction with the enzyme equals the nUmber of monomeric molecules in the solution. 2) Above the CMC, where part of the lipid is present as F“; 29 micelles rather than as monomers, the values on the X-axis, while equalling the total concentration, do not represent the true concentration of the substrate as related to its interaction with the enzyme. MULTIPLICITY OF PROTEINS The term isozyme, as loosely defined, refers to different molecular forms of an enzyme serving the same or a closely related function (125). This definition is general and connotes no specific type of structural relationship between the protein species which may be observed to have similar enzymatic activities. The methods by which isozymes may be generated have been classified into three convenient categories by Harris (126). Multipleggene loci coding structurally distinct polypeptide ghains of a protein. In one case nonidentical polypeptides may be associated in various members of a set of isozymes, so that the individual isozymes vary in the combination of PolyPeptides they possess. One example of this is the five laCtic dehydrogenase isozymes in which the A and B poly- PePtides form a tetramer series (127). Alternatively, poly- Pfiptide products of different loci may separately form the various members of a set of isozymes. This appears to be the case with phosphoglucomutase, for which there are three distinct loci, at each of which multiple alleles occur, giving rise to a series of isozymes and extensive heterogeneity of the protein (128,129). 30 Multiple alleles at a single locus. Since heterozygotes carry two different alleles they may be expected to show a more complex pattern of isozymes than homozygotes. An example of this is again seen with phosphoglucomutase, in which individuals homozygous at all three loci show at least eight isozymes, whereas heterozygosity at all three loci (‘3 can lead to as many as fifteen recognizable forms (130). Secondary modifications of protein structure. The first two categories are further complicated by structural changes which modify the assembled protein. These may involve deamination of glutamine or asparagine, phosphorylation of serine residues, addition of carbohydrate groups, or removal of components from the protein by proteolytic enzymes. Structural variants of this nature have been observed with phosphoglucomutase (130), adenylate kinase (131), adenosine deaminase (129), and peptidase B (129). Four compilations (132-135) of proteins which have been detected in multiple forms in man have been published. The Primary proteins, which occur as multiple forms in leukocytes, erythrocytes or plasma, have been summarized in Table 2 using these primary sources. This table is far from complete and does not include many of the blood proteins and clotting faCtors in plasma which have been found to occur in more than one form. When the Jacob-Monod model of regulation of gene activity in the lac system of E. coZi was first proposed there If W11. 31 Table 2. Enzymes Having Multiple Forms in Human Blood Protein No. of Forms Haptoglobin a-chain B-chain Transferrin Pseudocholinesterase Albumin Glucose-6-phosphate dehydrogenase Carbonic anhydrase Erythrocyte esterase Acid phosphatase Catalase 6-Phosphate gluconate dehydrogenase Myoglobin Phosphoglucomutase PGM I PGM II PGM III Fibrinogen Adenylate kinase Lactate dehydrogenase A subunit B subunit Amylase al-Acid glycoprotein B-Lipoprotein Galactose-l-phosphate uridyl transferase Malate dehydrogenase (soluble) Malate dehydrogenase (mito.) 01 Antitrypsine Glutathione reductase Ceruloplasmin Peptidase A Peptidase B HYpoxanthine-guanine phosphoribosyl transferase Glutamic oxalacetic transaminase NADH di aphoras e PhOsphohexose isomerase 10 ...: com NVMU‘INU'IU'IVU'I #LNNU'IGJ ONNO‘ #AuNmNNNNNhi—‘m H 32 were several applications of this concept to human structural and control genes in an attempt to explain the multiplicity of observed proteins (136-139). However in most cases in which this hypothesis has been tested it has been found to be fallacious (132,140-144). Data on the primary structure of the human enzymes are not obtainable in most cases, thereby making it impossible to make a distinction between allelism and genes at different loci. MATERIALS AND METHODS MATERIALS Sources of Enzymatic Activity Human Plasma: Blood from voluntary donors was collected in either heparin or EDTA containing a sodium cation. The unrefrigerated blood was centrifuged at full speed in an IEC clinical centrifuge and the plasma was decanted and stored at 4°. Alternatively, out-dated plasma, containing acid- citrate-dextrose (ACD), was obtained from the American Red Cross (Lansing, Mich.). Fabry plasmas, obtained from blood collected in either heparin or EDTA with a sodium cation, were the generous gifts of Dr. William Krivit, Dr. Michel Philippart and Dr. Matthew Spence. Human Kidney: Human kidney, excised from a lS-year old male accident victim approximately 2 hr following death, was obtained through the courtesy of Dr. William Walker of the Pathology Department of St. Lawrence Hospital (Lansing, Mich.). Human Urine: Urine was collected without refrigeration and concentrated lS-fold using an Amicon hollow fiber ultrafiltration system having a molecular weight cut-off of 50,000. 33 ( ’l ‘x 34 Cohn Fractions: Cohn fractions IV-l and I were obtained through the courtesy of Dr. James Sgouris of the Michigan Department of Health (Lansing, Mich.). Alternatively ceramide trihexosidase (Form A) was purified from an acetone precipitate of Cohn fraction IV-l prepared by the National Red Cross (Bethesda, Md.). Reagents The common reagents used were all of reagent-grade quality. The special reagents used are listed below. Solvents General Solvents All solvents were redistilled by constant flow rotary evaporation. Dry Methanol Methanol was dried by distillation from magnesium turnings containing a catalytic amount of iodine and was stored over molecular sieves. Dry Pyridine Pyridine was dried by distillation from barium oxide after refluxing 4 hr and was maintained over Drierite. Resins Affinose 201, Bio-Rad Laboratories, Affinose 202, and Richmond, Ca. Bio~Gel A-Sm Silicic Acid Clarkson Chemical Co., (Unisil) Williamsport, Pa. Sephadex G-10 Pharmacia, Uppsala, Sweden Amberlite‘s CG-120, Mallinckrodt, CG-400, and IR-120 McGraw Park, II. 35 Dowex 50W-X8 Darco G-60 Celite Chromatography Supplies Silica Gel-G Plates Kieselguhr G OV-101 and SE-30 Silylating Reagents Bis(trimethylsilyl)- trifluoroacetamide Pyridine-Hexamethyl- disilazane-Trimethyl- chlorosilane Electrophoretic Supplies Ampholine Carrier Ampholytes Sepraphore III Polyacetate strips High Resolution Buffer (Tris-barbital-sodium barbital) Coomassie Blue, Ponceau S, Nitro Blue Tetrazolium, and Phenazine Methosulphate Schiff's Reagent J. T. Baker, Phillipsburg, N.J. Fisher Scientific Co., Fair Lawn, N.J. Johns-Manville, Denver, Co. Quantum Industries, Fairfield, N.J. Macherey, Nagel and Co., Duren, Germany Applied Science Laboratories, Inc., State College, Pa. Regis Chemical Co., Chicago, Il. Prepared according to Sweeley et al. (145). LKB, Rockville, Md. Gelman Instrument Co., Ann Arbor, Mi. Gelman Instrument Co., Ann Arbor, Mi. Sigma Chemical Co., St. Louis, Mo. Scientific Products (Harleco), Detroit, Mi. 36 Detergents Triton X-100 Bile Salt Detergents Enzymes Galactose Dehydrogenase Galactose Oxidase and Catalase Phospholipase A2, Invertase, and Neuraminidase Scintillation Fluid DPO Toluene Aquasol Radioactive Chemicals [1-14C]Stearic Acid (54 mCi/mmole) [14C]UDP-N-Acety1- glucosamine (40 mCi/mmole) [14C]CMP-Sialic Acid (1.3 x 106 dpm/umole) Rohm G Haas Philadelphia, Pa. Sigma Chemical Co., St. Louis, Mo. Boehringer Mannheim, New York, N.Y. Worthington Biochemical Corp., Freehold, N.J. Sigma Chemical Co., St. Louis, Mo. Prepared by dissolving 50 mg POPOP and 4.0 gm PPO per liter of toluene. POPOP and PPO are supplied by Packard Instrument Company, Inc., Downers Grove, I1. New England Nuclear, Boston, Ma. New England Nuclear Boston, Ma. New England Nuclear Boston, Ma. Dr. Saul Roseman, Johns-Hopkins University 37 Miscellaneous Chemicals Phospholipids N-(p-Nitropheny1)- oxamic Acid p-Nitr0pheny1-a- galactoside 4-Methylumbelliferyl- a-galactoside l-Ethyl-3(3-dimethyl- aminopropy1)carbodiimide p-Nitrophenol, Sodium hydrosulfite, Sodium methylate, and Anthrone Hydrogen Bromide Polyethylene Glycol 6000 Melibiose, Stachyose, and Tyrosine Serdary Research Laboratories, London, Ontario K G K Laboratories, Jamaica, N.Y. Pierce Chemical Co., Rockford, Il. Koch-Light Laboratories Ltd., Colnbrook, England Ott Chemical Co., Muskegon, Mi. Fisher Scientific Co., Pittsburgh, Pa. Matheson Gas Products, Chicago, Il. Matheson, Coleman 8 Bell Detroit, Mi. Sigma Chemical Co., St. Louis, Mo. All other miscellaneous cofactors, enzymes and reagents were purchased from Sigma Chemical Co. 38 METHODS Purification of Lipids Isolation of GL-3 and GL-Zb: GL-3 and GL-2b were isolated from formalin-fixed Fabry kidneys. Total lipids were extracted using a modification of the original Folch Procedure (146). The minced kidney tissue was homogenized with seven volumes of methanol (w/v) in a Sorvall Omni-Mixer at room temperature. Sufficient chloroform was added to make the solvent ratio of chloroform-methanol 2:1, after which the mixture was homogenized a second time. The cellular debris was removed by filtration using a Buchner funnel containing coarse-grade solvent-washed filter paper. The residue was extracted with 10 volumes (based on the original weight) of chloroform-methanol (2:1). A volume of 0.1 M KCl equivalent to one-fifth that of the final volume of the combined extracts was added. The solvents were mixed and allowed to stand at 4° until the two phases completely separated. The washed lower phase from the Folch extraction was evaporated to dryness under reduced pressure and the residue was extracted with 200 ml of acetone. The precipitate was removed by filtration and washed with 200 ml of diethyl ether. The residue of crude glycolipids obtained from the acetone and ether extracts was subjected to mild alkaline hydrolysis according to the method of Vance and Sweeley (147). The residue obtained from mild alkaline hydrolysis was taken up in chloroform-methanol (19:1) and applied to a 250 gm silicic I. G -..'II_J~'o H.” Itt 39 acid column prepared in chloroform-methanol (19:1). The individual glycolipids were eluted stepwise with chloroform- methanol solutions consisting of 12%, 14%, 16%, 20%, 30%, and 50% methanol. Each solvent was applied until the Land's spot test was negative (148). The volume of each fraction was reduced in vacuo by constant flow rotary evaporation and an aliquot was streaked on a pre-coated, non-heat activated silica gel G plate and chromatographed in chloroform-methanol-water (100:42:6). Fractions containing pure glycolipids were evaporated to dryness. The residue was dissolved in benzene-chloroform (2:1, v/v), frozen in liquid nitrogen and lyOphilized into a white powder. The purity of the glycolipids obtained by this method was assessed by gas-liquid chromatography, following methanolysis, as described by Vance and Sweeley (147). Isolation of GL-2a: Lactosylceramide was isolated from porcine erythrocyte stroma. The stroma were prepared from 20 liters of porcine blood, laked by addition of glacial acetic acid to a final concentration of 5%, as described by Yamakawa (149). The extraction and isolation of lipids from the stroma followed the procedure outlined above. Preparation of Phospholipids: Commercially prepared PhOSpholipids were tested for purity using the two dimensional thin-layer system of Rouser et al. (150). Phospholipid (20 ug) was applied to a non-heat-activated silica gel G Plate and chromatographed in chloroform-methanol-28% aqueous 40 ammonia (6S:25:5). The plate was air-dried for 10 min and chromatographed in chloroform-acetone-methanol-acetic acid- water (3:4:1:l:0.5). A single spot was taken as an indication that the lipid was at least 95% pure and no further purification was performed. When several lipids were present in a sample, the desired phospholipid was purified from the 1’71 mixture using silicic acid chromatography as outlined by Sweeley (151). Chemical Modification of GL-3 F ‘1“ Preparation of [SHlGL-3: GL-3 (10 umole) was suspended in 10 ml of 0.01 M potassium phosphate buffer, pH 7.0, containing 0.02 M sodium cholate. Galactose oxidase (2 mg) and catalase (1 mg) were added and the solution was incubated at 37° overnight. Sodium borotritiide (20 umole; 140 uCi/umole) was added slowly at room temperature and allowed to stand with occasional mixing for 8 hr. The mixture was evaporated to dryness under nitrogen and the residue was taken up in chloroform-methanol (19:1). The sample was applied to a silicic acid column and eluted with 12% methanol in chloroform followed by 25% methanol in chloroform. The residue obtained by evaporation of the 25% eluate was dissolved in chloroform-methanol (2:1, v/v) and continuously dialyzed against running tap water for 6 days to remove exchangeable hydrogen atoms. 41 Preparation of Galactosyl(al+4)galactosyl(81+4)- glucopyranose: Trisaccharide was obtained from GL-3 by a modification of the Wiegandt procedure (152). GL-3 (100 mg) was dissolved in 25 m1 of dry methanol. Ozone, produced by a Welsbach ozonator, was introduced at room temperature until ozone consumption ceased, as determined by a positive reaction when a filter paper saturated with a KI-starch solution was introduced into the flask. The solution was evaporated to dryness under reduced pressure at 60°. The residue was dissolved in 10 ml of 0.2 M sodium carbonate and allowed to react for 12 hr at 20°. After neutralizing the solution with Dowex 50W>X8 (H+), it was extracted three times with 10 ml portions of hexane to remove the fatty acids. The trisaccharide was obtained by lyOphilization of the lower aqueous phase obtained from the hexane extractions. Preparation of GL-2a by Chemical Degradation of GL-3: GL-2a was prepared by a modification of Taketomi's procedure for preparation of lysohematoside (153). GL-3 (100 mg) was added to 10 ml of 90% aqueous butanol containing a final concentration of 1 N KOH. The mixture was placed in a sealed tube and heated at 80° for 8 hr. After cooling to room temperature, the reaction mixture was dialyzed against constant running tap water for 2 days. The glycolipid mixture was evaporated to dryness under reduced pressure at 80°. The residue was dissolved in chloroform and applied to a silicic acid column prepared in chloroform. FLA ".'fiu’-- : 42 Lactosylceramide was eluted with 12% methanol in chloroform. Enzymatic Syntheses Preparation of Galactosy1(o1+6)galactosyl(al+6)- glucopyranose: Stachyose (2.5 gm) was dissolved in 100 ml of 0.01 M sodium acetate buffer, pH 4.8. Invertase (5 mg) was added and the mixture was incubated at 27° for 12 hr. The concentrated reaction mixture (10 ml) was applied to a column (5.3 i.d. x 45 cm) composed of a prewashed mixture of Darco G-60 and Celite (equal parts by weight). Mono- saccharide, trisaccharide and unreacted stachyose were sequentially eluted from the column with water, 15% ethanol and 20% ethanol, respectively, as described by Whistler and Durso (154). Ethanol was removed from the 15% eluate by rotary evaporation and the trisaccharide was obtained from the water solution by lyOphilization. Preparation of Lysolecithin: Lysolecithin was prepared enzymatically by the action of phospholipase A2, from Crotalue adamanteus venom, on lecithin as described by Wells and Hanahan (155). Lysolecithin was separated from unreacted lecithin by silicic acid chromatography. Preparation of (14C1GL-3 Preparation of Trihexosylsphiggosine: [14C]GL-3 was prepared by condensation of N-dichloroacetyl-3—0-benzoy1- sphingosine with acetobromotrisaccharide to form trihexosyl- l4 sphingosine. C-Labeled fatty acids were coupled to the 43 trihexosylsphingosine using 1-ethy1-3(3-dimethylaminopropy1)- carbodiimide. Trisaccharide (50 mg), obtained by ozonolysis of GL-3, was added to 4 ml acetic anhydride-dry pyridine (1:1, v/v). The suspension was heated at 80° until the carbohydrate dissolved, then allowed to stand at room temperature for F" 12 hr. Dry methanol (5 ml) was added and the mixture was allowed to stand for 2 hr. The solution was evaporated to dryness under reduced pressure at 60° and the product was dried in vacuo over potassium hydroxide pellets. The acetylated trisaccharide was brominated by a modification of the procedure outlined by Wolfrom and Thompson (156). The trisaccharide (35 mg) was dissolved in 10 ml of a freshly prepared solution of acetic anhydride containing 40% hydrogen bromide. The mixture was allowed to stand at room temperature for 20 min before extraction with two 10 m1 portions of chloroform. The combined chloro- form extracts were evaporated to dryness under reduced pressure at 30°. Final traces of acetic acid were removed by drying in vacuo over potassium hydroxide pellets. N-Dichloroacetyl-3-0-benzoylsphingosine was prepared and coupled to the acetobromotrisaccharide (25 mg) using mercuric cyanide as outlined by Shapiro et al. (157). The protecting groups were removed by 0.5% sodium methoxide treatment followed by 5% barium hydroxide at 80° for 1 hr as described by Shapiro et al. (157). The reaction mixture 44 was dialyzed against constant running tap water overnight and the dialyzed solution was evaporated to dryness in vacuo. CQEPl}E£.9f Trihexosylsphingosine and (14C]Fatty Acids: Trihexosylsphingosine (5 mg), dissolved in 1 ml dichloro- methane-methanol (1:1, v/v), was added to a solution containing 5.0 mg of [1-14C]stearic acid (54 mCi/mmole) and 5 mg 1-ethyl-3(3-dimethylamin0pr0pyl)carbodiimide dissolved in 1 ml dichloromethane-methanol (1:1). Acetonitrile (1 ml) was added and the mixture was incubated at 40° for 12 hr. The incubation mixture was evaporated to dryness in vacuo and the residue was dissolved in 5 ml of chloroform. The chloroform solution was washed successively with two 1 ml portions of 0.1 M sodium bicarbonate, 0.1 N HCl, and water, then dialyzed against constant running tap water for 8 hr. The [14C]GL-3 was evaporated to dryness under reduced pressure and the residue was dissolved in water containing 0.03 M sodium taurocholate. Preparation of a-Galactosidase Affinity Column Adsorbent Melibiose Octaacetate: elibiose octaacetate was prepared and brominated by modification of the procedures described by Wolfrom and Thompson (156). Acetic anhydride (50 ml) and dry pyridine (50 ml) were added to 5 gm of melibiose. The suspension was heated at 80° until the carbohydrate dissolved, then allowed to stand at room temperature for 12 hr. Dry methanol (100 ml) was added slowly 45 with stirring and allowed to stand for 3 hr. The solution was evaporated to dryness under reduced pressure at 60° and the product was dried in vacuo over potassium hydroxide pellets. Acetobromomelibiose: Melibiose octaacetate (8.8 gm) was dissolved in 50 ml of a freshly prepared solution of acetic anhydride containing 40% hydrogen bromide. The mixture was allowed to stand 45 min at room temperature, then was filtered 3 :"n through a sintered glass funnel into 150 m1 chloroform. The chloroform solution was washed twice with 70 ml portions of water, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure at 30° until the chloroform was removed, then at 60° to remove acetic acid. Final traces of acetic acid were removed by drying in vacuo over potassium hydroxide pellets. pritrgphenylacetomelibioside: Coupling of p-nitrOphenol to acetobromomelibiose was based on the Koenigs-Knorr reaction for Oligosaccharide synthesis (158). Acetobromomelibiose (4.8 gm) was dissolved in acetone (25 ml) and added to 2.0 gm p-nitrophenol dissolved in 25 ml 1.0 M sodium carbonate. The mixture stood at room temperature overnight and was evaporated to dryness under reduced pressure at 60°. The residue was taken up in chloroform (400 m1) and extracted with 100 ml IPOItions of 0.25 M glycine-carbonate buffer, pH 9.5, until no lyellow color was obtained in the aqueous phase. The chloro- formIlayer was evaporated under reduced pressure at 30°. 46 Deacetylation: The product of the coupling reaction (4.2 gm) was suspended in 300 ml dry methanol containing 0.5% sodium methylate (w/v). Following gentle stirring at room temperature for 26 hr, the precipitated materials were removed by filtration and discarded. The supernate was neutralized by stirring with Amberlite IR-120 (H+) and the neutralized F“ solution was evaporated under reduced pressure at 60°. harm-v.5... ‘ . -1 Reduction of p-Nitrophenylmelibioside: p-Nitrophenyl- melibioside (1.6 gm) was dissolved with sonication in 100 ml of distilled water containing 5% methanol. The pH was “ adjusted to 7.4 with 0.01 N sodium hydroxide after which sodium hydrosulfite (5 gm) was added with stirring. The solution was stirred for 10 min, desalted by passage through a column of Sephadex G-10, and evaporated to dryness under reduced pressure at 60°. Coupling_of p-Aminophenylmelibioside to Succinoyln aminoalkyl-Agarose based on the Method of Cuatrecasas (159): Affinose 202 (10 ml packed bed) was washed with 20 bed volumes of 0.1 M sodium chloride, pH 6.0. p-Aminophenylmelibioside (1.6 gm), dissolved in 150 m1 of 40% dimethylformamide, was added to the washed resin suSpended in 50 ml of 40% dimethyl- formamide. After adjusting to pH 5.0 with 0.1 N HCl, a solution of l-ethyl-3(3-dimethylamin0propyl)carbodiimide (1 gm) in 8 m1 of water was added over a 10 min period. The reaction was allowed to proceed at room temperature for 24 hr 47 with occasional shaking. The product of the coupling reaction was packed into a column and washed successively with 40% dimethylformamide, water and 0.001 M MES buffer, pH 5.4, until UV-absorbing materials and carbohydrate [as measured by the anthrone reaction (160)] were absent from the eluate. Thin-layer Chromatography of Carbohydrate Derivatives: Kieselguhr G plates of 250 u thickness were prepared by the method of Lewis and Smith (161). The plates were heat- activated at 80° for 2 hr prior to use in either of the following solvent systems: Solvent 1: Benzene-ethyl acetate (1:1, v/v) Solvent II: Butanol-pyridine-water (75:15:10, v/v) Melibiose octaacetate, acetobromomelibiose and p-nitrophenyl- acetomelibioside were chromatographed in Solvent I using paper-lined tanks which were pre-equilibrated for a minimum of 6 hr. p-Nitrophenylmelibioside and p-aminOphenylmelibioside were chromatographed in Solvent 11 using paper-lined tanks which were pre-equilibrated for a minimum of 12 hr. Identification of Products by GLC: An aliquot (50 ul) of the solution obtained from Sephadex chromatography of p— aminophenylmelibioside was evaporated to dryness under nitrogen. The residue was silylated with 20 ul of bis(tri- methylsilyl)trifluoroacetamide-dimethylformamide (1:1, v/v) and analyzed with a Perkin-Elmer 9000 gas chromatograph equipped with a 3 ft x 1/8 in i.d. column, packed with 0.05% 48 OV-lOl on textured glass beads (Corning Glass). The oven temperature was increased linearly from 140° to 300° at 10°/min. The residue from a second 50 ul aliquot was dissolved in 3 ml of 0.5 N methanolic HCl and heated at 80° for 24 hr. The methyl glycosides were silylated with 20 ul of pyridine- hexamethyldisilazane-trimethylchlorosilane (10:4:2) (145) and analyzed with a Hewlett-Packard 402 gas chromatograph equipped with a 6 ft x 1/8 in i.d., 3% SE-30 column, maintained at 165°. Operating Conditions: A standard procedure was used for affinity chromatography of all samples. After equilibrating the column (0.6 cm i.d. x 6.5 cm) and sample to the Optimal pH for a specific enzyme, the sample, not exceeding 100 ml in volume, was percolated through the column until it reached the top of the resin, then the flow was stOpped for 30 min. The column was eluted with the buffer used for sample application until the non-adsorbed proteins were eluted (20-30 fractions depending upon the sample volume); then 0.1% Triton X-100 (v/v) was added to the eluting buffer. All fractions (1 ml) were eluted at 4° using the maximum flow rate of the column, kept constant by use of an LKB peristaltic pump. Specific samples were prepared for affinity chromatography as follows. Urine and plasma were adjusted to pH 5.4, using 0.1 M citric acid, prior to chromatography. Partially purified ceramide trihexosidases, Form A, from an— 49 plasma and kidney were dialyzed against 0.001 M MES buffer, pH 5.4, containing 5% butanol. Partially purified ceramide trihexosidases, Form B, were dialyzed against 0.01 M sodium phosphate buffer, pH 7.2, containing 5% butanol. Ficin was dissolved in 0.05 M sodium acetate buffer, pH 4.5, and applied to the affinity column after it had been equilibrated with the same buffer. Proteins without a-galactosidase activity were dissolved in 0.001 M MES buffer and chromatographed in the same manner as the ceramide trihexo- sidases. Preparation of Neuraminidase Affinity Column Couplipg:pf Tyrosine to Affinose 201: Affinose 201 (10 ml packed bed) was washed with 20 bed volumes of 0.1 M sodium chloride, pH 6.0. The washed resin was suspended in 200 ml of 40% dimethylformamide containing 30 mg of tyrosine. After adjusting the solution to pH 5.0, with 0.1 N HCl, 2 gm of l-ethyl-3(3-dimethy1aminopropy1)carbodiimide, dissolved in 10 ml of water, was added with gentle stirring over a 10 min period. The reaction was allowed to proceed at room temperature for 24 hr with occasional shaking. The product of the coupling reaction was packed into a column and washed with distilled water until uncoupled tyrosine was absent from the eluate, as determined by spectrOphotometric measurements at 280 nm. IIJT—— w A .- _ 50 Reduction of N-(p-Nitrophenyl)oxamic acid: N-(p-Nitro- phenyl)oxamic acid (5 mg) was dispersed in 100 m1 of 5% methanol by sonication. After adjusting to pH 7.4 with 0.01 N sodium hydroxide, 5 gm of sodium hydrosulfite was added with rapid stirring. The solution was stirred for 10 min, acidified with 0.1 N HCl, filtered, and evaporated to dryness. Copplingpofpp-Aminophenyloxamic Acid to Tyr—Succinoyl- aminoalkyl-Agarose as Described as Cuatrecasas and Illiano (162): p-Aminophenyloxamic acid (3 mg) was dissolved in 5 ml of cold 0.4 N HCl, and 30 mg of sodium nitrite, dissolved in 1 ml of water, was added with striring over a one-minute period. After stirring an additional 5 min, the mixture was added to the tyr-Affinose 201, suspended in 25 ml of 0.5 M sodium bicarbonate buffer, pH 8.9. The pH was readjusted to 8.8 and the reaction was allowed to proceed for 8 hr at room temperature with gentle stirring. The coupled resin was washed with 4 liters of 0.1 N sodium chloride. Operating Conditions: Commercially prepared Clostridium perfringene neuraminidase, Type VI, was chromatographed essentially as described by Cuatrecasas and Illiano (162). Neuraminidase (30 mg) was dissolved in 7 m1 of 0.05 M sodium acetate buffer, pH 5.5, and dialyzed for 12 hr at 4° against four liters of 0.05 M sodium acetate buffer, pH 5.5, containing 2 mM CaCl2 and 0.2 mM EDTA. The dialyzed material was applied to the column (0.6 x 5 cm), equilibrated with the same buffer. The column was eluted with 0.1 M NaHCOS, pH 9.1. 51 The eluted fractions (1 ml) were adjusted to pH 6.0 with l N NaOH and tested for neuraminidase activity by incubation with porcine submaxillary mucin. Assays for a-Galactosidase Activity Assay for Ceramide Trihexosidase Activity: Method 1 (Incubation of GL-3 with Crude Enzyme Preparations): Crude preparations include whole plasma, concentrated urine, and all samples obtained from steps prior to affinity chromatography. With these samples the incubation consisted of the following: 0.5 ml of crude enzyme preparation; 0.1 ml bovine serum albumin (5 mg); 0.2 umole of GL-3 dissolved in 0.1 ml of water containing 0.96 mg sodium cholate; and buffer to a final volume of 1.0 m1. Assays for ceramide trihexo- sidase, Form A activity, were buffered with 0.2 M citrate- phosphate (sodium cation), pH 5.4, whereas those for ceramide trihexosidase, Form B activity, were buffered with 0.2 M sodium phosphate buffer, pH 7.2. Incubations without GL-3 served as controls. The mixture was incubated for 4 hr at 23°, and the reaction was then terminated by addition of 2 ml of chloro- form-methanol (2:1, v/v). The solution was vortexed and centrifuged at full speed in an IEC clinical centrifuge to separate the two phases. The upper phase was removed and clarified by boiling for 10 min, cooling in an ice bucket and recentrifuging. Enzymatic activity was detected by assaying the upper phase for liberated galactose. "I 52 Method 2 (Incubation of GL-3 with Purified Enzyme Preparations): The incubation consisted of the following: 7 1.68 x 10- M ceramide trihexosidase Form A-l; 0.2 umole GL-3 dissolved in 0.1 m1 of water containing 0.96 mg sodium cholate; 0.03 M sodium taurocholate (0.04 M sodium taurocholate if assaying ceramide trihexosidases, Form A-2); 0.15 M sodium chloride; and either citrate-phosphate or sodium phosphate buffer to a total volume of 0.5 m1. Assays with boiled ceramide trihexosidase served as controls. After 4 hr incubation at 23° the reaction was terminated by addition of 1 m1 of chloroform-methanol (2:1). The upper phase was separated and clarified as described in Method 1. Method 3 (Incubation of Purified Ceramide Trihexosidase with Radioactive Substrate): The incubation consisted of 7 the following: 1.68 x 10' M ceramide trihexosidase Form A—l; 0.2 umole unlabeled GL-3 dissolved in 0.1 m1 of water containing 0.96 mg sodium cholate; 1.4 x 10'4 umole of [14C]GL-3 (10,000 cpm) or 2.7 x 10'3 umole [3H]GL-3 (22,000 Cpm); 0.03 M sodium taurocholate (0.04 M for ceramide trihexo- sidase, Form A-Z); 0.15 M sodium chloride; and either citrate- phosphate or sodium phosphate buffer to a total volume of 0.5 ml. Assays with boiled ceramide trihexosidase served as controls. After 1-4 hr incubation at 23° the reaction was terminated as described in Method 2. Radioactivity in [14C]lactosylceramide or [3H]galactose was determined as described in the following section. 53 Method 4 (Assay in Butanol): A solution of ceramide trihexosidase, Form A-l, (1 volume) was mixed with one volume of butanol. After thorough mixing, the butanol phase was decanted and the aqueous phase was washed with 1 volume of butanol. The butanol extracts were combined and an aliquot (1 ml) was removed for determination of enzymatic activity. Incubations contained 1.4 x 10.4 umole of [14C]GL-3, 0.2 umole GL-3, 0.03 M sodium taurocholate, and 0.1 N NaOH to pH 5.4, in a total volume of 1.5 ml. Assays with boiled ceramide trihexosidase served as controls. The mixture was incubated 4 hr at 23° then terminated by boiling for 30 min. After centrifugation the reaction mixture was evaporated to dryness. The residue was taken up in chloroform-methanol (2:1) and dialyzed overnight against constant running tap [14 water. C]GL-2a was determined as described in the following section. Method 5 (Incubation of Trisaccharide with Ceramide Trihexosidase): The incubation consisted of the following: 7 1.68 x 10' M ceramide trihexosidase; 0.2 umole tri- saccharide [galactosyl(al+4)galactosyl(81+4)glucose]; 4 mg lecithin; 5 mg bovine serum albumin; and citrate-phOSphate buffer, pH 5.4, to a final volume of 0.5 ml. After 2 hr incubation at 23° the reaction was terminated by addition of 1 ml of chloroform-methanol (2:1). The upper phase was separated and clarified as described in Method 1. 54 Methods for Detecting the Hydrolysis Products: [Spectrpphotometric Quantitation of Liberated Galactose]: Galactose liberated from unlabeled GL-3 was determined with an end-point assay consisting of 77 ul of 0.1 M tris buffer, pH 8.6; 20 01 NAD+, (10 mg/ml) Sigma Grade v; 100 01 of upper phase; and 3 ul of galactose dehydrogenase added in 1 M (NH4)ZSO4; in a final volume of 0.2 ml. The increase in absorbance at 340 nm was measured with a Gilford 2400 Model recording spectrOphotometer thermostated at 30°. An absorbance change of 0.01 was equivalent to 0.32 nmoles of galactose. From the observed absorbance change, corrected for the control without added GL-3, galactose in the entire upper phase from the incubation was calculated. Qpantitation of Liberated Galactose by Gas-Liquid ChromatOgraphy: Alternatively, liberated galactose was quantitated by gas-liquid chromatography. The upper phase obtained from the enzyme incubation (Method 1) was passed through a column (0.5 cm i.d. x 4 cm) of mixed bed resin composed of equal quantities of Amberlites CG-120 (H+) and CG-400 (OH-). Sugars were eluted from the column with 5 ml of methanol-water (1:1). The eluates were evaporated to dryness and the residue was dissolved in approximately 100 01 of methanol-water (9:1). These solutions, alternated with standard galactose solutions, were applied to Whatman No. l chromatography paper. The carbohydrates were separated by chromatography in isoprOpanol-acetic acid-water (3:1:1) 55 (163). The sections spotted with standard galactose were removed from the chromatogram and visualized with aniline- acid-oxalate (163). The areas adjacent to the standard galactose were eluted from the chromatogram in the following manner: the paper was placed in 2 ml of methanol-water (1:1) and allowed to stand at room temperature for 1 hr, after which the solution was heated to boiling, cooled, and the paper removed and rinsed with 1 ml of methanol-water (1:1). 3 - 1.12 x 10'2 umole, based on the Mannitol (2.8 x 10' quantity of residue obtained from the column eluates) was added to these solutions as an internal standard. The samples were evaporated to dryness and 5 ul of bis(trimethyl- silyl)trifluoroacetamide-dimethylformamide (1:1, v/v) was added to the residue. After standing for one-half hour at 80° the samples were analyzed with a Hewlett-Packard 402 gas chromatograph equipped with a 6 ft x 1/8 in i.d. column packed with 3% SE-30 and maintained at 170°. The yield of galactose (in nmoles) was calculated from the GLC data by the method of Vance and Sweeley (147), using a correction factor of 1.2 to account for the differences in the relative molar response of the detector to TMSi mannitol and TMSi galactose. The data obtained from a method control and the enzyme incubation controls were used to correct this value. v) S6 Detection of [6-3HlGalactose: The reaction was terminated with chloroform-methanol and separated into two phases as previously described. After removing the upper phase, the lower phase was washed twice with 1 ml portions of theoretical upper phase. The combined upper phases were washed once with 1 ml of lower phase. The upper phases were desalted as described in the preceding section and reduced to a constant volume of 0.5 m1. An aliquot of this solution (100 01) was spotted on Whatman #540 filter paper (2.1 cm circle), the paper was dried, and placed in 10 ml of DPO toluene. Alternatively the entire 0.5 m1 sample was added to 15 m1 of Aquasol for counting. Radioactivity in these samples was monitored in a Beckman LS-150 liquid scintillation counter. Controls with boiled ceramide tri- hexosidase were treated in an identical manner. Detection of Liberated [14C]GL-2a: The incubation was terminated by vortexing with 1 m1 of chloroform-methanol (2:1). After separating the two phases by centrifugation, the upper phase was washed twice with 1 m1 of chloroform. The combined lower phases were evaporated under nitrogen, the residue was dissolved in approximately 0.2 m1 of chloro- form-methanol (2:1), and the solutions were transferred to pre-coated silica gel G plates. The glycolipids were separated by chromatography in chloroform-methanol-water (100:42:6). Chromatography tanks were paper-lined and equilibrated for a minimum of 8 hr prior to use. Tanks were 57 used to develop two plates simultaneously and discarded. The area of [14C]GL-2a corresponding to standard lactosyl- ceramide was scraped directly into 10 m1 of DPO toluene and counted in a Beckman LS-lSO liquid scintillation counter. Controls containing boiled ceramide trihexosidase were treated in an identical manner. Assay for Digalactosylceramide : Galactosyl Hydrolase: The standard reaction mixture for the assay of digalactosyl- ceramidase (in the dialyzed acetone precipitate obtained from Cohn fraction IV-l) consisted of 0.5 ml of enzyme solution, 0.2 umole of GL-Zb dissolved in 0.5 mg aqueous sodium cholate, 0.03 M sodium taurocholate, 0.15 M sodium chloride and citrate-phosphate buffer, pH 5.5, in a final volume of 1.0 ml. Following partial purification by affinity chromatography or isoelectric focusing, 0.2 ml of enzyme solution was incubated as above in a total volume of 0.5 m1. Incubations containing boiled enzyme served as controls. The reaction was terminated with chloroform-methanol (2x the volume of the incubation) and the two phases were separated as previously described. Liberated galactose was determined spectrophotometrically, using galactose dehydro- genase, as described in the preceding section (Method 1). Assays for Non-specific a-Galactosidases 4-Methy1umbellifery1-O-ga1actoside as Substrate: The standard reaction mixture contained 100 pl of enzyme solution, 2.2 nmoles of substrate and 0.1 M citrate-phosphate buffer, 58 pH 4.5, to a constant volume of 0.5 ml. After incubation at 370 for 2 hr the reaction was terminated by addition of 2.5 m1 of 0.1 M ethylenediamine buffer, pH 11.2. The mixture was centrifuged and the fluorescence of the liberated methyl- umbelliferone was measured using a Turner fluorimeter with 365 nm excitation and 450 nm fluorescence filters. Boiled enzyme controls were used. p-Nitrophenyl-a-Galactoside as Substrate: Enzyme solution (100 01) was added to 0.5 ml of 0.1 M citrate- phosphate buffer, pH 3.0, containing 2.0 pmoles of substrate. After 2 hrs at 370 the reaction was terminated by addition of 0.1 ml of 0.01 N sodium hydroxide and 0.5 m1 of 0.2 M glycine-carbonate buffer, pH 9.5. The absorbance was read at 420 nm using a Gilford 2400 Model spectrophotometer. Boiled enzyme controls were used. Plasma Infusions Plasma for infusion was obtained by plasmapheresis of freshly drawn, heparinized blood from cross-matched normal donors whose previously assayed plasma had normal concentrations of ceramide trihexosidase activity (Method 1). A 17-year old hemizygote received 550 ml of plasma (2145 units)’ over a 30 min period. A 31-year old hemizygote received 600 ml of plasma (4680 units)* over a 30 min period. *A unit of ceramide trihexosidase activity is defined as the amount that liberates 1 nanomole of galactose per hour at pH 7.2 and 23 C. 59 In a third experiment blood was drawn from two normal donors and processed through normal blood bank procedure at the American Red Cross. The heparinized plasma (540 ml containing 2584 units)* was administered to a 30-year old hemizygote over a 1 hr period. At two hour intervals post-infusion blood was obtained f A by venepuncture or through the aid of a heparin lock for '- enzymatic and/or substrate determinations. — is .—-.Z. .- ‘P - Isolation of Ceramide Trihexosidases Purification of Human Plasma Ceramide Trihexosidases, Form A Extraction: The enzymes were isolated from Cohn fraction IV-l, prepared by Method 6 of the low temperature ethanol precipitation described by Cohn et al. (164). Enzymatic activity was preferentially extracted from Cohn fraction IV-l by dispersing the frozen protein paste (200 gm) in 600 m1 0.001 M MES buffer, pH 5.4, using a Waring blendor at low speed. After removal of the undissolved material by centrifugation at 16,000 g for 30 min at 4°, the supernatant solution was adjusted to pH 7.0 with 0.01 N sodium hydroxide. Ammonium Sulfate Treatment: To the 750 m1 of buffered supernate, maintained at 4°, 487 gm of ammonium sulfate (0 to 80% saturation) was added with stirring over a period *A unit of ceramide trihexosidase activity is defined as the amount that liberates l nanomole of galactose per hour at pH 7.2 and 23°C. _ 60 of 1.5 to 2 hr. After 30 min of additional stirring, the suspension was centrifuged for 30 min at 16,000 g and the precipitate was discarded. Butanol Treatment and Acetone Precipitation: To 990 m1 of the 80% ammonium sulfate supernate, maintained at 4°, 52 ml of n-butanol was added slowly with stirring over a 1 hr period as described by Morton (165). The 5% butanol solution was stirred for an additional hour at 4° then transported to a -20° room. Following the method of Askonas (166), 1,563 ml of acetone, chilled to -20°, was added slowly with stirring so as to make the final concentration 60% acetone. The protein-ammonium sulfate precipitate was removed immediately from the acetone solution by suction filtration. The precipitate was slurried with about 30 ml of 0.001 M MES buffer, pH 5.4, containing 5% butanol and dialyzed against 100 volumes of the same solution for 8 to 12 hours. Affinity Chromatoggaphy: Following dialysis, the purification was completed by affinity chromatography as previously described. Pilot-Scale Isolation of Ceramide Trihexosidases, Form A: The following protocol was submitted to the National Red Cross for preparation of ceramide trihexosidases, Form A: 61 Dissolving the material (pH 7.0; temperature should not exceed 4°): Cohn fraction IV-l (10 kg) is dissolved in 9 volumes (90 l) of 0.25 M sodium phosphate buffer, pH 7.0. Any undissolved material should be removed by centrifugation at 700 g or above. Ammonium sulfate treatment (pH 7.0; temperature should not exceed 4°): The SUpernate is adjusted to 80% saturation with solid ammonium sulfate (50.6 kg or 111 lbs). Precipitated proteins are removed by centrifugation (10,400 g) and discarded. Butanol treatment (pH is unadjusted; temperature should not exceed 4°): Butanol (5 l) is added to the 80% ammonium sulfate supernate with stirring. It is desirable to stir the solution for at least one hour following the addition of butanol. Acetone precipitation (pH is unadjusted; temperature is -20°): Acetone (158 1) is added with stirring until the solution contains 60% acetone by volume. The resulting precipitate, consisting of a protein-ammonium sulfate sludge, is removed by either filtration or centrifugation immediately following the addition of acetone. The precipitate is stored at 0° or below. 62 Isolation of Ceramide Trihexosidase, Form B Extraction: Enzymatic activity was extracted from Cohn fraction 1, prepared by Method 6 of the low temperature ethanol precipitation described by Cohn (164). The enzymes were extracted from frozen Cohn fraction I by slowly stirring the protein paste in six volumes (w/v) of 0.01 M sodium phosphate buffer, pH 7.2, at room temperature for 30 minutes. The undissolved fibrinogen was removed by centrifugation at 16,000 g at 4° for 30 minutes. Removal of <:<>ntaminating proteins by ammonium sulfate precipitation, butanol treatment, acetone precipitation, and affinity chromatography were performed as previously described for Forms A. The separation of the multiple forms of ceramide trihexosidase, Form B, was completed by isoelectric focusing. Isolation of Ceramide Trihexosidase from Human Kidney Extraction: The kidney of a lS-year old male accident ViCt in: was excised approximately two hours following death. The outer capsule was removed and the tissue was minced by Passage through a meat grinder. The minced tissue (58.7 gm) was suspended in 150 m1 of 0.25 M sucrose. Homogenization was performed in a Waring blendor for six 30-sec periods at full speed, with intermittent cooling in an ice bath for 3‘5 minutes. The homogenate was centrifuged at 600 g for 30 min at 4° and the precipitate discarded. The supernate was Poured through cheesecloth to remove floating fat and centrifuged at 16,000 g for 30 minutes. 63 Release of Enzymatic Activity from the Particulate Fraction: The crude lysosomal-mitochondrial pellet (16,000 g precipitate) was suspended in 100 ml water (one-half the original volume of supernate) and treated with sodium cholate (10 mg/ml) overnight, as described by Brady et al. (32) - The sodium cholate-treated solution was then centrifuged at 100,000 g for 90 min and the precipitate dis c arded. Concentration of the Enzymatic Activity: To the 110 m1 of 100,000 g supernate, 56.8 gm of ammonium sulfate (0 to 80% saturation) was added with stirring over a 1 hr period. Stirring was continued for an additional 30 min and the precipitated proteins were removed by centrifugation at 16,000 g for 30 min and discarded. To the 136 m1 of 80% ammonium sulfate supernate, maintained at 4°, 7 ml of butanol was added slowly with stirring over a 1 hr period as described by Morton (165) . Stirring was continued at 4° Overnight. Ceramide trihexosidase activity was Precipitated from the butanol solution by addition of acetone to a final concentration of 60% as described for the plasma enzymes. The ammonium sulfate-protein precipitate was di‘311Y2ed and chromatographed on an affinity column in the manner described for the plasma ceramide trihexosidases, Form A . 64 Miscellaneous Assays Protein Determinations: Protein was determined during enzyme purifications by the method of Lowry et al. (167) as modified by Hess and Lewin (168) . For affinity column profiles protein was measured with commercial Biuret reagent as follows: protein solution (0.1 ml) and 0.2 m1 of Biuret reagent were mixed and allowed to stand at room temperature for 30 min. The absorbance was read at 540 nm using a Gilford 2400 Model spectrOphotometer. Bovine serum albumin was used to prepare standard curves. Sialic Acid Determinations: Free sialic acid was determined by the periodate-resorcinol method of Jourdian et al . (169). N—Acetylneuraminic acid was used to prepare a standard curve. Mglecular Weight Determinations §ucrose Density Gradient Centrif_ugation: Sucrose dens ity gradient studies were conducted by the method of Martin and Ames (170), using a Beckman SW 50 rotor at 50.000 rpm at 0° for 14 hr. Linear gradients from 5-20% sucrose without added preservatives were employed. The Pretein (8 ug) was applied to the gradient in 0.2 m1 of 0°001 M MES buffer. Fractions (0.15 ml) were collected and assayed for ceramide trihexosidase activity, while the Positions of the marker enzymes, cytochrome c and hemoglobin, were determined by spectrOphotometric measurements at 550 nm. (IL. I ‘0. 65 Gel Chromappgraphy: The ceramide trihexosidases were individually chromatographed on a Bio-Gel A-Sm column (3 x 95 cm) along with cytochrome c, B-lactoglobulin, leucine aminopeptidase, hemoglobin, and glyceraldehyde-3-phosphate dehydrogenase (1 mg each) as standards (171). The column was equilibrated with 0.01 M MES buffer, pH 5.4, and the proteins were applied and eluted with the same buffer. Elut ed proteins were detected by Spectrophotometric measure- ments at 280 nm. The same series of proteins was chromatographed on another column of Bio-Gel A-Sm (3 x 95 cm) equilibrated with 0.01 M tris-chloride, 1% SDS, pH 8.0 and 2% 2-mercapto- ethanol (172). The proteins were dissolved in 5 m1 of this solvent, sonicated briefly, and heated at 80° for 30 min. The proteins (1 mg each) were applied to the column and eluted with the same buffer. Eluted proteins were detected in the following manner (168): an aliquot (4 ml) of each fraction was mixed with 4 m1 of 5% TCA and allowed to stand for 15 min at 0° when centrifuged. The aqueous solution was removed and 0.2 m1 of 0.5 N NaOH was added to the sample. This sample was mixed with 0.4 m1 of commercial Biuret reagent and protein was determined as previously described. Egtrgwhoretic Methods isoelectric Focusing: Isoelectric Focusing was performed essentially as described by Vesterberg and Svensson (173). The Sucrose density gradient, with 50% sucrose (W/V) 35 the 66 densest solution, was prepared and layered into a 110 ml LKB electrolysis column, as suggested by the manufacturer. The column was maintained at 1° with a thermostated Lauda K-ZR water bath. Wide pH ranges were equilibrated for 48 hr with a maximum potential of 600 V, whereas narrow pH ranges were equilibrated for 72 hr with a maximum potential of 700 V. After focusing of the carrier ampholytes, fractions (0. S or 2.0 ml) were eluted from the column keeping the flow rate constant with a peristaltic pump. The pH of the individual fractions was determined using a Corning Model 12 pH meter with the electrode equilibrated at 20°. The fractions were assayed for enzymatic activity and/or protein without removal of the ampholytes. Polyacrylamide ElectrOphoresis: Polyacrylamide electro- phoresis in 9% gels was performed by the method of Fairbanks et al. (174). The gels were scanned at 280 run, then stained With Coomassie Blue. Following destaining with 10% TCA-33% methanol by the method of Johnson (175) the gels were scanned at 550 nm. Cellulose Acetate Electrophoresis: Protein solution (2 111 containing 1-4 08 Protein) was streaked onto a buffer- damPened Sepraphore III polyacetate strip. ElectrOphoresis was Carried out in 0.05 M tris barbital-sodium barbital buffer, pH 8.8, with a current of 0.5 mA/cm strip width for 30‘40 min. 67 Staining of Activity: The cellulose strip was overlaid with a filter paper saturated with a solution containing 4 x 10"4 M unlabeled substrate (CL-3 or GL-Zb), 0.03 M sodium taurocholate and 0.15 M sodium chloride dissolved in either 0.001 M MES buffer, pH 5.4, or 0.01 M sodium phosphate buffer, pH 7.2. This reaction was kept moist for 4 hr at 37°. When Form A and Form B proteins were on the same strip, it was stained for 2 hr at pH 7.2 and for 2 hr at pH 5.4. The strip was then Sprayed three times, at five minute intervals, with a fine mist of a solution containing 20 ul of galactose dehydrogenase and 5 mg NAD+ in 1.0 m1 tris buffer, pH 8.6. The strip was then overlaid with a filter paper saturated with tris buffer, pH 9.2, containing 6 x 10'5 M Nitro Blue Tetrazolium and 3.3 x 10'5 M phenazine methosulphate. Staining was carried out at 37° for 1 hr in the dark. Stainipgpof Glycpproteins: The cellulose strips were immersed in 5% TCA for 2-3 min to precipitate the proteins. They were then rinsed in tap water and transferred to Muller's colloidal iron oxide reagent (176) for 60 min, washed in 3% acetic acid until excess ferric chloride was removed, as determined by an absence of color upon addition of potassium ferrocyanide, then immersed in a 2% solution of potassium ferrocyanide in 2% HCl for 30 min. The strip was washed with distilled water and transferred to 1% periodate for 30 min, then to commercial Schiff's reagent for 30-60 min, following thorough rinsing with distilled 68 water. Following the develOpment of reddish-purple bands, the strips were washed in three changes of 0.5% sodium metabisulfite (5 min each) and dried. Ponceau S Staining_forpguantitation of Proteins: The cellulose strips were stained with 0.2% Ponceau S made in 3% aqueous trichloroacetic acid for 20-30 min then destained in 5% acetic acid. The strips were sectioned between protein bands and the individual segments were added to 0.1 N NaOH (0.5-1.0 ml depending upon the intensity of the stain) and the dye was eluted by vortexing for about 2 min. The absorbance at 565 nm, read against a blank prepared from an unstained section of the strip, was measured with a Gilford 2400 spectrophotometer. The protein level was determined from the absorbance by comparison with a standard curve obtained by electrophoresis, staining and elution of the dye from 1-10 ug of albumin. Interconversion of Ceramide Trihexosidases, Forms A and B Neuraminidase Treatment of Partially Purified Ceramide Trihexosidases, Form A: An extract (40 m1) of Cohn fraction IV-l, containing 7 mg of protein per ml, was made in 0.5 M citrate-phosphate buffer, pH 5.4. Commercially prepared neuraminidase (14 mg) was added and the mixture was incubated for 3 hr at 37°. Following incubation, samples (1 ml) were assayed for ceramide trihexosidase activity over the pH range 4.6 to 8.0 69 In a second experiment whole plasma (35 ml) was adjusted to pH 5.4 with 0.5 M citrate-phosphate buffer, pH 3.0. The diluted plasma, containing 70 mg of protein per ml, was mixed with 125 mg of neuraminidase and incubated at 23° for 4 hr. At one-hour intervals samples (1 ml) were assayed for enzymatic activity at pH 5.4 and 7.2, and separate samples (0.5 ml) were used to determine sialic acid liberated. Controls, in which neuraminidase was omitted, were treated under identical conditions. Treatment of Purified Plasma Ceramide Trihexosidase (Form A-l) with Neuraminidase: Purified plasma ceramide trihexosidase, Form A-l, 300 pg in 4 m1 of 0.001 M MES buffer, pH 5.4, was added to 16 m1 of 0.2 M citrate-phosphate buffer, pH 5.4. Purified CZ. perfringens neuraminidase, Type VI (50 units)* was added and the mixture was incubated in a Dubnoff shaking incubator at 37°. Aliquots (5 ml) were removed after 1 and 4 hr and were concentrated by dialysis against polyethylene glycol 6000 in preparation for cellulose acetate electrOphoresis. After 2 hr incubation 10 ml was removed and dialyzed against 0.001 M MES buffer, then concentrated by dialysis against polyethylene glycol 6000. This sample was used for isoelectric focusing in the pH range 3-10. Two controls were run, one in which the * A unit of neuraminidase is defined as that quantity which will liberate 1.0 umole of N-acetylneuraminic acid per minute at pH 5.4 and 37° using porcine submaxillary mucin as substrate. 70 neuraminidase was omitted from the incubation and one in which the incubation containing neuraminidase was maintained at 00 for 4 hr. Incorporation of [}4C1UDP-N-Acetylglucosamine into Ceramide Trihexosidase, Form B: Ceramide trihexosidase, Form B-V (pI 8.4), was partially purified from Cohn fraction I and was separated from the other B forms of the enzyme by a combination of affinity chromatography and isoelectric focusing. Enzyme B-V (600 ug) in 5 ml of 0.001 M MES buffer, pH 5.4, was added with gentle stirring to 50 mg of porcine kidney homogenized in 20 m1 of 0.25 M sucrose, pH 7.0. [14C]UDP-N-Acetylglucosamine (10 umole), 20 umole ATP, 20 umole phosphoenolpyruvate, and 20 umole CTP were added and the mixture was incubated in a Dubnoff shaking incubator at 370. After 4 hr incubation, the reaction mixture was centrifuged at 12,000 g for 30 min. The precipitate was washed twice with 5 m1 portions of 0.001 M MES buffer, pH 5.4, containing 15% butanol. These extracts were combined with the 12,000 g supernate and concentrated by dialysis against polyethylene glycol 6000. The concentrated protein solutions (285 pg) were divided into 3 equal aliquots and electrophoresed on cellulose acetate strips. The cellulose strips were treated as follows: one was stained with Ponceau S and used for protein determinations; one was stained for ceramide trihexosidase 71 activity; and one was sectioned and added directly to 10 ml of DPO toluene for counting in a Beckman LS-150 liquid scintillation counter. Three controls were run, one in which the basic protein was eliminated from the incubation mixture; one in which [14C]UDP-N-acetylglucosamine was omitted from the incubation; and one in which [14C]UDP-N-acetylglucosamine, ATP, CTP, phosphoenolpyruvate, and ceramide trihexosidase were incubated in the absence of kidney homogenate. Incorporation of [14C]CMP-Sialic Acid into Ceramide Trihexosidase, Form B-V: The above experiment was repeated using [14C]CMP-sialic acid instead of [14C]UDP-N-acetyl- glucosamine. The only exceptions in the procedure were the following: 1) the [14C]CMP-sialic acid (10 umole) was added in 4 aliquots at one hour intervals and 2) the cofactors ATP, phosphoenolpyruvate and CTP were eliminated. hill- or I.‘ ‘2‘ 'l 1 : :I %"h RESULTS IDENTIFICATION OF RADIOACTIVE SUBSTRATES Preparation of [3HJGL-3 _ . . . i GL-3 purified from Fabry kidney was treated With 5 ' galactose oxidase from Dactylium dendroidee to oxidize the g terminal galactose residue to galactohexodialdose (177). Following sodium borotritiide reduction and silicic acid chromatography the product was chromatographed on a silica gel G plate in chloroform-methanol-water (100:42:6). The plate was scanned for radioactivity after which the standard GL-3 was located by staining the lipid with iodine vapors. As shown in Figure 3, the majority of the radio- activity co-chromatographed with standard GL-3, although a small amount of radioactive material migrated slightly ahead of GL-3. The radioactive compound was eluted from the silica gel and added to carrier GL-3. After methanolysis and conversion of the carbohydrates to trimethylsilyl derivatives, GLC analysis, shown in Figure 4, proved that the compound con- tained galactose and glucose in a molar ratio of 2:1. The carbohydrate, fatty acid and sphingosine moieties obtained by methanolysis of [3H]GL-3 were counted and found to contain 75.8%, 11.1% and 13.3%, respectively, of the total 72 73 .oumam ecu mo one some :0 wouuoam momOAOSmmueH. wcwewmwcoo mam. Hothe may kn :mom oflhum may spa: woemflam mm: m-40 eumecmum mo :Oflufimom one mafiZOLm oumam 049 on» mo ucwhaosan oah .fiouweuooav poum3-Hocm:poE -anwoaofico mafia: oumaa 0 How wowaflm m :o eonamemoumEOAno mm: m-40H:m. m-au.:m. mo eeom datum .m oasmAa 74 75 Figure 4. GLC of TMSi Methyl Glycosides from [3H]GL-3 The methyl glycosides were silylated with 20 ul of pyridine-hexamethyldisilazane-trimethylchlorosilane (10:4:2) and analyzed with a Hewlett-Packard 402 gas chromatograph equipped with a 6 foot 3% SE-30 column maintained at 165°. 76 Ban-.11 12 Omcoamoa 5.00.00 Minutes 77 radioactivity. Glucose contained 3.4% of the radioactivity associated with the carbohydrate, as determined by counting the methyl glycosides recovered from the flame detector during GLC analysis. The [3H]GL-3, obtained in 85% yield, contained 22,000 cpm/2.7 x 10'3 umole. Preparation ofii4C]GL-3 Attempts were made to prepare trihexosylsphingosine by deacylating GL-3 using the alkaline hydrolysis procedures of Taketomi (153) and Carter (178). The Carter method, requiring a 12 hour reflux in 50% aqueous dioxane containing 5% Ba(OHJZ, produced nine unidentified degradation products and no detectable trihexosylsphingosine. The milder procedure of Taketomi, which requires heating the lipid in 90% aqueous butanol containing 1 N KOH, produced 70% GL-Za and 0.5% 'trihexosylsphingosine. Therefore it was necessary to synthesize trihexosylsphingosine using Shapiro's method for preparation of psychosine (157) . Triliexosylsphingosine was identified by thin-layer chromatography and GLC analysis. The product chromatographed With an Rf of 0.10-0.13 on silica gel G plates developed in chloroformtmethanol-4N ammonium hydroxide (60:40:9). .After nmthanolysis of this compound, the mixture of carbohydrates was converted to trimethylsilyl derivatives and analyzed by gas chromatography. The results afforded evidence for the Presence of galactose and glucose in a molar ratio of 2:1. 78 The product obtained by coupling of [14C]stearic acid and trihexosylsphingosine was chromatographed on a silica gel G plate using chloroform-methanol-water (100:42:6) and the plate was scanned for radioactivity after drying. The majority of the radioactivity co-chromatographed with standard GL-3, as shown in Figure 5. [14C]GL-3, having 4 umole, was obtained in 5% yield, 10,000 cpm/1.4 x 10- based on the quantity of sphingosine used as starting material. PREPARATION OF a-GALACTOSIDASE AFFINITY COLUMN Choice of Carbohydrate for Coppling to Affinose 202 Since it was not feasible to synthesize enough trihexosylSphingosine to be used to prepare an affinity column having a sphingosine-containing ligand, analogous to those prepared by Sloan et al. (179) for the purification of sphingomyelinase and glucosyl ceramide hydrolase, a substrate analog of GL-3 was required. The ability of enzymes normally hydrolyzing substrates solubilized in micelles to cleave water-soluble analogs of the lipophilic molecule in a "pseudo-micellar" system was recently reported by Gatt (124). Thus the ability of the ceramide trihexosidases to cleave oligosaccharides was investigated. As shown in Figure 6 the partially purified ceramide trihexosidases, Form A, hydrolyzed a tetra— saccharide (stachyose) and several trisaccharides [galactosy1(al+6)galactosyl(al+6)glucose; galactosyl(al+4)- “W113 ‘ it... _ 79 .oumHa ego mo 0:0 some so wouuoam onume ecu xn zoom mflhum one cues eocwfifim mm: m-40 eumwcwpm mo :owufimom ecu mcwzonm oumaa 049 on“ mo unflMQOSHn 02H .moumenooav noumz-ao:mcpoe . -Epomoeofino wean: oumam 0 How mofiafim n no vocamnwoumEopno mm: n-40H04H m-so.oea. 0o doom datum .m oaswaa 80 81 .4 Show .mommefimoxocflhu oefiEmHoo mammaa 0:0 an omoxgomum mo mfimxH090>E ofiumexucm Am. .OpmhumESm mm Hoganhomfivtam.HamOpomHmmm4+H00meouumHmm Ho omoosfim -notaavaxmouomfimmmoraovmeouomHmm ponufio mean: mam»a0h0>g oefihmgoommflhu pom oocflmuno who: muHDmOH HmofiueoeH .4 Snow .mommefimoxogfihp 00HEono mammaa one an n-4r. omownwaos 0:0 m-o-v omoonamn4+amvamepomHmmm4+HovH>mouomHmm mo mamkaOhezz m4. moumhumnsm ooflhmnoummomflao wo mfim%HOh0>m oflpmexuem .o thmflm 82 a a <—° e °° (IN/P910194" :06 snow") £04009 ouowfizuz: |.O (D, O .— e 0 .5 0' -.‘.=. g s 0' 8’ N O m 9: 5i, i7 it. to o :w/pamequ :06 Salem") Wuov 000me Q 0.6 0.8 mg Lecithin / ml 04 OZ 83 galactosyl(81+4)-glucose; and galactosyl(al+4)galactosyl- (Bl+4)-sorbitol] equally well at optimal concentrations of phosphatidylcholine, while hydrolysis of the disaccharide (melibiose) was negligible. Although melibiose was not a good substrate for the enzyme, the coupling of p—nitrophenol from which an amine ..-1 could be produced for attaching the carbohydrate to Affinose 202 would result in a compound having approximately the same size as a trisaccharide. In addition, the 14 A spacer attached to the Affinose 202 might confer some of the preperties of GL-3 to this product. Therefore melibiose was chosen as the carbohydrate for coupling to the affinity column. Product Identification The chemical synthesis of the affinity column adsorbent, the structure of which is shown in Figure 7, was followed primarily by thin-layer chromatography. The Rf value for the product of each reaction is shown in Table 3. In addition, melibiose octaacetate was identified by mass spectrometry using an LKB 9000 mass spectrometer and the p-nitrophenyl and p-aminophenyl derivatives were analyzed by GLC both before and after hydrolysis with methanolic HCl. Prior to hydrolysis the trimethylsilyl derivatives of p- nitrophenylmelibioside and p-aminophenylmelibioside had the same retention time as a standard trisaccharide [galactosyl- (al+6)-galactosyl(al+6)-g1ucose] when analyzed by gas . 84 uconhom04 meadow zuflcfimm4 omwwflmouomamo-d .n oeswflm :o I ..o I z .1 szo.12a.~xo.zzo~.~:o.ozz o ... m A... .1 .1 I o: o N... ..ofo 89.004 86 Table 3. Rf of Carbohydrate Derivatives All carbohydrate derivatives were chromatographed on Kieselguhr G plates heat-activated at 80° for 2 hr prior to use. Solvent I is benzene-ethyl acetate (1:1, v/v) and Solvent II is butanol-pyridine—water (75:15:10, v/v). Compound Solvent Rf Melibiose Octaacetate I 0.51 Acetobromomelibiose I 0.63 p-Nitrophenylacetomelibioside II 0.71 p-Nitrophenylmelibioside II 0.44 p-Aminophenylmelibioside II 0.60 87 chromatography on a 0.05% OV-lOl column. Following acid hydrolysis and conversion of the carbohydrate mixture to trimethylsilyl derivatives, GLC analysis proved that the molar ratio of glucose and galactose was 1:1. The attachment of the p-aminOphenylmelibioside to Affinose 202, containing 8 umole carboxyl group content per ml packed bed, was estimated to be 6-7 nmoles per m1 of packed resin, from the amount of uncoupled material obtained from the coupling reaction and by thorough washing of the packed resin. The adsorbent prepared by this method can be used repeatedly without apparent loss of binding capacity. However the flow rate decreased after 45-50 runs and could not be improved by repacking the resin. Specificity for a-Galactosidases Investigations were carried out to determine if the substituted Affinose 202 selectively adsorbed only a- galactosidases. The proteins selected for this purpose were bovine serum albumin, B-lactoglobulin, mucin, trans- ferrin, and liver B-galactosidase. These studies, summarized in Table 4, proved that the substituted Affinose was not a non-specific protein adsorbent. Adsorption of Ceramide Trihexosidase To insure that the attached carbohydrate was responsible for adsorption of the ceramide trihexosidases, a partially purified fraction of ceramide trihexosidases, Form A, isolated as described in the following section, was 88 I'— Table 4. Specificity of a-Galactosidase Affinity Column k All proteins were applied to the affinity column in 9 10 m1 of 0.001 M MES buffer, pH 5.4. Chromatography was i performed using the conditions described in Materials and Methods. . . Mg Recovered as Prote1n Mg Applied Non-adsorbed Protein Bovine Serum Albumin 100 99.0 B-Lactoglobulin 100 98.0 Mucin 100 98.7 Transferrin 100 100.0 B-Galactosidase (liver) 100 99.0 89 chromatographed on a column of unsubstituted Affinose 202. Both the protein and enzymatic activity emerged together in the break-through protein as shown in Figure 8. No additional activity or protein could be eluted from the unsubstituted resin when detergent was added to the buffer. The recovery of enzymatic activity from the unsubstituted Affinose was consistently in excess of 97%. AFFINITY CHROMATOGRAPHY OF NEURAMINIDASE Synthesis of Affinity_Column Adsorbent The affinity column adsorbent for purification of neuraminidase, shown in Figure 9, was analogous to that described by Cautrecasas (162), differing only in the spacer used for attaching the ligand to the matrix backbone. The coupling of tyrosine to Affinose 201, having 8 umole carboxyl group content and an 8 A spacer, was estimated to be 7-8 nmoles per m1 of packed bed, while the coupling of N-(p- nitrOphenyl)-oxamic acid was estimated to be 6 nmoles per m1 of packed bed. Both estimates were based on the amount of unreacted material recovered from the coupling reaction. The adsorbent prepared by this method was unstable, lasting for approximately 5 runs. Purification of Neuraminidase Affinity chromatography of commercially purified CZoetridium perfringens neuraminidase consistently gave 40-45 fold purification of the enzyme. Rechromatography 9O .xufl>flpom ommefimoxocfihu oefismpoo 0:0 :wouoea How eozmmmm mm: m00wpompw as H ozu mo zoom .meoguoz 0:0 mflmfinoumz cw confluomoe mm eozamemoumEoezo mm: H->H :oauompe 0:00 anw eohmmopm oumuflmfloohm oeouoom wenxfimfiw one mom omoeflww4 voyeufiumnsmcz co Ommwflmoxonflhe oeflsmho0 mo wagon 4 may we xzmmhwoamEOch xpficwwm4 .m ohswfim 91 (+) ugalmd bu: a 9 8+8. 8. [—D s a a. s' to V‘ N (—<>—) 9924:0494: 2:19 salowu IOO IO Effluent (ml ) 92 peonpomo4 :ESHO0 xpflcfimw4 ommoflcflEmHSoz .m ohsmflm 93 IooowIz O l ...O / \z.z_ / \ azozwrzwaroazoxz :08 0 009.004 94 of the purified enzyme did not result in either increased purification or loss of specific activity. A typical purification is summarized in Table 5. DETERMINATION OF PRODUCTS LIBERATED BY ENZYMATIC HYDROLYSIS OF GL-3 Enzymatic Determination of Liberated Galactose : Ceramide trihexosidase activity was determined routinely by quantitation of the galactose liberated, using an end- point assay with galactose dehydrogenase (Method 1). This 2": J $ ~ I q assay was linear over the range of 0.56 nmole-560 nmoles of galactose, using a total assay volume of 0.2 ml. The reproducibility of the assay for 0.56-5.6 nmoles of galactose was f2.5%. Determination of Radioactive Hydrolysis Products [3H]GL-3 and [14C]GL-3 were prepared to enable rapid determination of the liberated hydrolysis products by a method which would be less affected by organic solvents and other possible inhibitors than the galactose dehydrogenase assay. The determination of radioactive products released by ceramide trihexosidase (Method 3) cannot be used with crude preparations which have a high enough protein concentration to form a protein pellet between the chloroform and aqueous phases upon termination of the incubation. In this case 75-90% of the radioactivity, depending on the quantity of protein, was bound to the protein pellet and the results were not reproducible. In addition [SHJGL-3 had a 95 Table 5. Purification of CZostridium perfringens Neuraminidase Neuraminidase (30 mg) was taken up in 7 ml of 0.05 M sodium acetate buffer, pH 5.5, and dialyzed for 12 hr at 4° against 0.05 M sodium acetate buffer, pH 5.5, containing 2 mM Pfi_ CaClz and 0.2 mM EDTA. The dialyzed material was applied to F the affinity column.(0.6 x 5 cm) equilibrated with the same buffer. Enzymatic activity was measured using porcine submaxillary mucin as substrate. Purification Step il‘ ‘4'. | '. Sample applied on column Protein (mg) 30 Volume (m1) 7 Specific activity* 3.6 Sample eluted from the column Protein (mg) 0.65 Specific activity* 160 Yield of Activity (%) 99 Fold Purification 44 * Specific activity = nmoles N-acetylneuraminic acid liberated per minute per mg of protein. 96 tendency to equilibrate between the aqueous and chloroform phases, necessitating extreme care to insure that all of the radioactivity in the upper phase was actually [3H]- galactose. OCCURRENCE OF CERAMIDE TRIHEXOSIDASE IN HUMAN PLASMA P Presence of Ceramide Trihexosidase Activity in Normal ( Plasma é Early attempts to determine whether added lysosomal ceramide trihexosidase from porcine liver and kidney would be active in human plasma at pH 7.0 led to the discovery of ceramide trihexosidase activity in normal human plasma. Aliquots (0.5 m1) of plasma were incubated for 4 hr with 100 nmoles of unlabeled GL-3, and liberated galactose was determined by an end-point assay with galactose dehydrogenase (Method 1). A bimodal curve of enzymatic activity, shown in Figure 10, indicated that there might be two forms of ceramide trihexosidase in plasma, with pH Optima at 5.4 and 7.2. Normal levels of enzymatic activity, summarized in Table 6, were about 8 nmoles of galactose liberated per ml of plasma per hour at pH 5.4 and 17 nmoles per m1 of plasma per hour at pH 7.2. ngramide Trihexosidase Activity in the Sphingolipidoses Fabry plasma was assayed to determine whether this glycosidase, presumably responsible for the enzymatic hydrolysis of GL-3, would be detectable. Plasma obtained ‘31» .2 _ 97 .N.0 000 0.4 00 0003000 000000 000 000000 000000000 -0000000 0003 000000000000 00 0000 00:00 0>0so 0000000 009 .o.0 0>000 0.20 000000 00 0000 003 .0.m :0 .000000 0009 .o.0 000 N.0 :0 0003000 0000 003 .m.e :0 .000000 002 2 0.0 0000003 am.0 000 0.4 :0 0003000 .o.m I0 .000000 000000000 -0000000 2 0.0 0003 00000000 003 :0 009 .000000 00003 0000 0000000 0 -X00000 00000000 00 0000000000000 ommmemzv 00000 00000000 00000000 0:0 00 000000 00003 000000 00000 0000 000 003 000000 :0 00 03030 00H 000000 000002 00 >00>00u4 0000000X00009 00000000 00 :0 mo 000000 .00 000M00 98 L l £12 0 (A417) lob salowrfiu J l o o m N A _ 1-4 99 Table 6. Ceramide Trihexosidase Activity in Human Plasma Enzymatic activity was determined using Method 1. Total units is expressed as nanomoles of galactose liberated per 4 hr per 0.5 ml plasma. Specific activity (S.A.) is expressed as nanomoles of galactose liberated per hr per mg of protein. pH 3}4 pH 7f2 SUbJeCt Total Units S.A. Total Units S.A. Fabry Hemizygotes A. G. <1.5 <0.01 onm mm weapom oEmm oz» mcflhsw «Emmfim :fl mHo>oH m-qo mEoupomv .N.n mm um mEmmHm mo HoyMHHHHHE pom use: pom m-qo Eopm wommofioh omouomamm mo moHoE: mm wommohmxo ma xufi>fluum uaumsxncm .flcofimsmcfl opp mo wco map um mm :mmum exp :0 ouv mammfim HmEuo: mo :oflmswcfi youwm omwomflw m.>Hnmm :ufiz mpcoflumm onE msomxuflao: 03p a“ xufl>flpom ommwflmoxonflup owflamhoo «Emmam Amoev «Emmfim oHogz mo :ofimswcH mcfizofiaom mammam xsnmm ca :oflpmuucoucoo oumhumnsm paw xuw>fiuo< oflumsxucm mo mHo>oq .HH ohsmflm 104 meson ovm com 09 ON. om o¢ o 1J1 _ a _ a _ L 1 _ _ _ o ...! - . . loom o o I. coo aw- . ‘ o . O n. w. mEEocmobaloolco J1| £11 1. o o ..O a I ‘0’ A U ¢ 7. 'A .o. w . 9 D dd I o m z m €88 ..lom m... . . $0 5. x w ... Table 7. 105 Concentration of Glycolipids in Fabry Plasma Following Infusion of Normal Plasma Glycolipids were quantitated by the method of Vance and Sweeley (147). A.G D.L. Time(hr) Volume (m1) GL-l GL-2 GL-3 GL—4 nmoles/100 m1 0 19.4 1.420 0.980 1.208 0.419 1 22.7 0.62 1.446 1.362 0.576 4 16.5 0.907 0.831 1.002 0.341 10 33.5 0.956 0.834 0.791 0.364 16.5 36.0 0.516 0.476 0.178 0.396 20 34.0 1.145 0.732 0.965 0.234 48 31.0 1.249 0.889 1.354 0.334 72 35.0 1.059 0.929 1.276 0.593 96 35.0 1.002 0.948 0.816 0.495 120 35.0 1.256 1.090 0.968 0.467 168 41.0 1.097 0.620 0.444 0.563 192 39.5 1.270 1.389 0.725 0.430 216 35.2 0.840 0.463 0.747 0.281 240 41.0 0.714 0.500 0.590 0.253 0 28.7 0.767 0.553 0.768 0.553 1 30.9 0.898 0.801 0.849 0.740 4 29.4 0.862 0.771 0.894 0.635 8 32.8 1.778 0.848 0.889 0.883 12 29.8 0.748 0.628 0.546 0.532 16 15.1 1.000 1.007 0.923 0.802 20.5 28.4 0.820 0.674 1.727 0.684 48 36.2 1.084 0.856 1.012 0.618 71.5 34.6 1.107 0.791 0.962 0.276 96 19.5 0.999 0.970 1.195 0.719 120 39.0 0.934 0.691 0.818 0.533 144 39.0 0.883 0.661 0.756 0.403 167.5 44.3 0.832 0.705 0.726 0.573 192.5 37.4 0.775 0.612 0.583 0.610 217 46.5 0.771 0.593 0.569 0.384 239.5 44.5 0.965 0.519 0.441 0.164 263.5 40.6 0.514 0.423 0.437 0.227 106 .mEmmHm mo HopflHflHHflE Hog use: you woN>H0prn m-qo mo moHoE: 3 :oflmsmcfl «Emmam mcfizoafiom m-o-v v.m In on xufi>fipum oflpmexuco :uflz wohmmEoo mfl mm pommohmxo ohm moflufi>fluum ofiumexuco :uom .flcofimswcw oz“ mo cam may um mH o .q.m xanmm msomeflEo; may :H fi-o-v N.n :m an xufi>fluow ommwfimoxonfihu owHEmhou mammflm HmEHoz mo :oflmswcH wcfizoaaom mammfim xpnmm a“ N.n :m paw v.m mm um xwfi>fipo< owumEchm mo acmfiHmQEou .NH ohswflm 107 luv/PGZAIOJPAH 2:19 $9IOUJU ,‘ l6 l4 l2 io Time (hr) 108 was coincident with the time periods of decreased ceramide trihexosidase activity at pH 7.2. Both enzymatic activities were slightly increased at 12 hours and their decay curves over the following 7 days were approximately parallel. Although ceramide trihexosidase activity appeared to be sporadic at both pH optima, summation of the individual activities, indicated that there were actually two increments of enzymatic activity without any periods of inactivation. In Vitro Studies: The apparent interconversion of the two forms of ceramide trihexosidase activity following plasma infusion was not an in vitro occurrence, since it could not be demonstrated by mixing donor plasma and Fabry plasma. Normal and Fabry plasma mixed in equal volumes and assayed without pre-incubation gave one-half the normal activity as expected. Pre—incubation of normal plasma containing 15.7 units of activity at pH 7.2 and 7.9 units of activity at pH 5.4 with an equal volume of Fabry plasma for 4 hours prior to the incubation for determination of enzymatic activity (Method 1) resulted in 26.7 units of activity at pH 7.2 and no detectable activity at pH 5.4, thus suggesting that at least in vitro, the enzymatic activity at pH 5.4 could be converted to that at pH 7.2 but not vice versa as suggested by the in vivo studies. 109 Comparison of Enzymatic Activity Determined by Artificial and Lipid Substrates Following Plasma Infusion A third patient was infused with normal plasma to study the difference in enzymatic activity toward artificial substrate and lipid substrate, as well as to insure that the enhancement of enzymatic activity following plasma infusion was not an artifact. A 30-year old hemizygote (R.R.) received 540 m1 of freshly prepared, heparinized plasma, containing 2584 units of enzymatic activity, over a 1 hour period. Enzymatic activity was determined by measuring the galactose liberated from GL-3 by both GLC and galactose dehydrogenase; an enhancement of ceramide trihexosidase activity occurred 3 and 8-12 hours post-infusion, as shown in Figure 13. The peak of optimal enzymatic activity occurred 6 hours post-infusion in the previous infusion experiments. Enzymatic activity measured with the artificial substrate, 4-methy1umbelliferyl-a-galactoside, shown in Figure 13, was completely inconsistent with the activity measured using the lipid substrate. With 4-methy1- umbelliferyl-a-galactoside there was an enhanced level of enzymatic activity immediately following infusion which sharply declined to pre-infusion levels within two hours post—infusion. ‘WJ '. 110 .m-o-v opfimouomamw -5-Hxhomflfiaonesfixcuoe-¢ Qua: muosdfifim oumhmmom co owma omHm who: mxmmmm mm-ozv xcmmpmoumEOH:o wflscfia-mwm kn paw n-4rv ommcomopwxnow omouomamw kn woumufipcmsc mm: omouomamw woumponflfi .m-qu spas mammfim mo muoscfifim HE m.o wo :ofipmnzocfl Houm< mEmmHm HmEHoz mo cowmswcH Hopmm ommomflm m.%unmm :pflz pcofiumm m cw xufi>fiuo< ommwmepumHmo-d «Emmfim .MH owsmfim 111 Jq/|w/sa|ou1u (flW-V) AllAllDV DIlVWAZNS '9 “i 09 V. '- '* 9 ° 1 l I l rh— ‘ - .L— . ‘ . Ir— ’a’ // ‘ —— ’x - I, r l J l V 00 N *0 O V (‘0 '- Slqy/lw/salowu (8-19) Ail/any oukuNa 12 10 TIME (hrs) 112 ISQLATION AND PURIFICATION OF CERAMIDE TRIHEXOSIDASES The results of plasma infusions suggested that enzyme replacement might have some therapeutic value in the treat- ment of Fabry's disease. Thus purification of ceramide trihexosidase was undertaken to make this enzyme available in a stable, concentrated form without large quantities of contaminants. Attempts were made to isolate ceramide trihexosidase activity from whole plasma, but the apparent instabilitycfi'the enzyme and the problem of acquiring fresh starting material resulted in negligible success. Separation of Plasma Ceramide Trihexosidase into Two Enzymatically Active Forms Out-dated plasma collected in 13% acid-citrate—dextrose (ACD) contained no enzymatic activity when assayed directly by the galactose dehydrogenase assay (Method 1). Protein concentrates from out-dated ACD plasma were prepared by the low temperature ethanol fractionation procedure described by Cohn at al. (164) and were tested for enzymatic activity. These fractions were active. As summarized in Table 8 Cohn fractionation of out-dated whole plasma separated the ceramide trihexosidase activity into two components. The component designated ceramide trihexosidase, Form A, had optimal activity at pH 5.4 and the second component, designated as ceramide trihexosidase, Form B, had Optimal activity at pH 7.2. Most of the Form A activity occurred in Fraction IV-l (by-product fraction) and 113 Table 8. Ceramide Trihexosidase Activity in Cohn Fractions The individual Cohn fractions (25 mg) were dissolved in 50 m1 of glass-distilled water. Each fraction (1 ml) was assayed for enzymatic activity using Method 1. The supernate of Fraction V was concentrated 50-fold by ultrafiltration prior to assaying for enzymatic activity. Fraction % of Total Specific Activity* Units/liter Plasma Plasma Protein pH 5.4 pH 7.2 pH 5.4 pH 7.2 II 5.1 ' <0.01 0.80 ----- 2,320 II + 111 26.7 <0.01 <0.01 ---------- IV - I 5.8 1.10 <0.01 3,520 ----- IV - 4 6.6 <0.01 <0.01 ---------- V 53.8 0.023 0.016 699 486 super. V 2.0 <0.01 <0.01 ---------- * Sp. Act. = nanomoles galactose liberated per mg protein per 4 hrs. 114 most of the Form B activity was recovered in Fraction I (fibrinogen fraction). Variable amounts of both forms of ceramide trihexosidase were found in Fraction V (serum albumin fraction), accounting for 15-20% of the total enzymatic activity. Similar binding of enzymatic activity could be demonstrated by adding bovine serum albumin to either Cohn fraction I or IV-l, thereby indicating that the enzymatic activities in Fraction V did not represent additional forms of ceramide trihexosidase. Isolation of Ceramide Trihexosidases Efforts at enzyme purification and characterization were concentrated on plasma ceramide trihexosidase, Form A, since it appeared to be metabolically the more significant of the two activities in view of the finding that the Fabry heterozygote retained this form of activity. Although primary emphasis was placed on the purification and characterization of plasma ceramide trihexosidase, Form A, the B form was also partially purified to investigate the relationship between the two forms of enzymatic activity. Kidney ceramide trihexosidase was partially purified for comparison with the plasma enzymes. Tables 9, 10 and 11 give the results for a typical purification of plasma Form A, plasma Form B, and kidney ceramide trihexosidases, respectively. The results are reproducible only if the conditions of the isolation and enzyme assays are stringently controlled, due to the anomalous effect of 115 omH mem.mm onm.~ mam Nv.o mahom < Mom fiance mHH coo.mm ~aa.~ mNo mN.o N-< shoe .n NA mam.am oo~.~ mam AH.o H-< show .m xnmmhwoum50hsu xuflcwmm< .HHH mmH mvo.fi . ma ONO.H m.mH Hosanna am as mfimxaafie .e AQH mfio.fi Ha omo.H m.4~ opmufiaflomha ocouoom woo .o o>fluomaw «.mm poumohu Hocmpsp am .n o>Huomcfl v.5m pamumCRQQDm aomNflamzv wow .m H->H do :ofiumcowuomhwpsm .HH OOH H no.9 amm owe.“ H->H coauomhm :nou .H 11c flasou-v mtgmmfimmwwmaco mtg\mofloeao Ames ampm th>ooom :oflumofiwflpsm onHwomm xufl>fiuu< awopOHm cowpmpflmflooum odouoom ou hofihm mfimxamfiw op oanmum xaamfiphmm xaco ohm paw mummasm anacoEEm wow swap oHoE mo venomoum may a“ o>fiuumafl osouon mommwwmoxonflhu mafiEmuou one < Show .mommwfimoxoawhe owfiEMHou «Emwam cmfisz mo :owumofiwflhsm .m manme 116 m.~omq¢ oma 0H.o >-m shoe .8 c.omm.m mom wo.o >H-m show .e o.ooo.m NmN Ao.o HHH-m seam .u m.mmH.H HmN oo.o HH-m Egon .H m.a~o.e mom HH.o H-m shoe .m 0Ha.mm ova w.oa~.v omo.N me.o mchsuoH uHHHuonomH .>H m.~om.a on“ OH.o m-m show .u m.mma.m com mH.o N-m show .H o.oa¢.¢ cos AH.o H-m spam .a 0Ha.mm cam w.oa~.¢ omo.~ ma.o anaauwoumsopnu suHcHHH< .HHH ooa.m man OAH Amm.H H.HH Hoamusn Hm .ma wouxamwu oumuwmwooum acououm woo . U m>HHuacH “.mN Hosanna Hm .n o>fiuomqw u.m~ oumcuomam aommHesz How .a H H0 nowumnoHuomnmnsm .HH H ocH mo.o 4¢~ Numa H :oHuoaHH :gou .H HeHoH-U Hag HHH\me\mmHoe:V Hhe\moHoe=V Hmav ampm :ofiumofimwusm xho>ooom xufi>fiuu< UHMHoomm xufi>fluo< :Hououm .cofiumuwmfioonm ocououa ou Howun mmeHmHv ou oHnmum AHHmenmm cho ohm use mummazm anacoasm wow :msu whoa mo oucomoum on» cw o>Huumcw oaooon mommvwmoxonfinu owfismnoo one m Show .ommvfimoxonwhe owflsmhou mamwam amen: mo newumofiwwhsm .oH magma 117 cmm.wN~ OHeH o.HamH o.oao HH.o seamhmoumeOggo AHHaHHH< .> Doom OHaH o.o0H o.oao a Hocapnn Hm .ma wouxamfiv .umm ocouoom woo .o ooa mHmH o.w 0.4Nw MOH emuamhp Hocmpsn Hm .B co mam ON.H o.aNH mOH a N a .Hmazm cm H :zv How .a WOH HHH Ho cowumcofluumRWn—Dm .>H mH VON om.o o.ONH Hoe moacHoasm m coo.OOH .HHH a VON mo.o m.Nm omHH mHmHHmHumHm m ooo.oH .HH H OOH No.0 H.ma woNN opacpmgsm w coo .H HeHom-V HHV Hea\me\mmHoe:U th\mmHoscv Hmev mofiumofimfipsm xho>oomm >9H>Huo< owmfloomm >uw>fiuo< :Hououm mouw < Show .ommwflmoxonfiuh ovHEmHou xocpfix cues: mo :oHumonHHDm .HH oHan 118 detergents, salt and enzyme concentration on the activity of the ceramide trihexosidases, as discussed in the following sections. Purification Steps Extraction of Ceramide Trihexosidase, Form A: The enzymatic activity was extracted from Cohn fraction IV-l using 0.001 M MES buffer, pH 5.4. It was also possible to extract the enzymes without loss of activity using buffer containing 5% butanol, however the butanol interfered with the ammonium sulfate step. The laboratory-scale procedure for selectively extracting ceramide trihexosidase activity was altered for the pilot- scale preparation. The Cohn fraction was completely dissolved in 9 volumes (minimum volume for dissolution) of 0.25 M sodium phosphate buffer, pH 7.0, to eliminate the necessity of removing a gelatinous precipitate and adjusting the pH prior to the ammonium sulfate treatment. The enzymes obtained by this procedure had no detectable differences from those isolated on a small scale. Extraction of Ceramide Trihexosidase, Form B: The ceramide trihexosidases, Form B, were extracted from Cohn fraction I by dissolving the frozen protein paste in sodium phosphate buffer at room temperature. Dissolving Cohn fraction I at 4° resulted in complete inactivation of the enzymatic acitivty. Otherwise the ceramide trihexosidases, Form B, were not noticeably cold-labile. 119 Extraction of Kidney Ceramide Trihexosidase: The time in performing the initial steps was critical since prolonged homogenization released Form A into the 16,000 g supernate where it has a half-life of 2-3 hours. The whole homogenate from human kidney contained approximately equal levels of the A and B forms of ceramide trihexosidase. During the preparation of the 16,000 g precipitate and sodium cholate treatment to release the enzymes from the membrane the activity of the B form disappeared and was accompanied by a concomitant increase in activity of Form A. Stabilization and Precipitation of the Enzymes: It was consistently observed that the addition of butanol stabilized both the A and B forms of the enzyme. Commonly employed stabilizers including 2-mercaptoethanol, Cleland's reagent, inert atmosphere and metal ions had no effect on the stability of the enzymes, while glycerol caused immediate inactivation of enzymatic activity. The temperature at which the butanol step is performed is not critical since addition of butanol to a final concentration of 5% without cooling does not depress the specific activity of the enzymes. The proteins were precipitated from the butanol solution by the addition of acetone to a final concentration of 60% following the low temperature method described by Askonas (166). The addition of acetone at 4° decreased the specific activity by one-half. After this step, which yields an ammonium sulfate-protein sludge containing 0.2% protein, 120 the enzymes can be stored as a frozen paste for at least six months. Although freezing causes total inactivation of the ceramide trihexosidases, enzymatic activity is completely restored by dialyzing the proteins overnight against 0.001 M MES buffer, pH 5.4, containing 5% butanol. After dialysis the ceramide trihexosidase, Form A, can be stored at 4° in 0.001 M MES buffer containing 5% butanol for one month without loss of activity, whereas Form B can be stored for two weeks without loss of activity under these conditions. The gradual loss of activity which occurs after this time is accompanied by the formation of a protein precipitate. Redissolving the precipitated protein restores 90% of Form A activity and 70% of Form B activity. Affinity Chromatographyyof Ceramide Trihexosidase, Form A: Since it had been determined that the ceramide trihexosidases hydrolyzed oligosaccharides in the presence of Optimal concentrations of phosphatidylcholine (lecithin), the possibility that lecithin would be required for the enzyme to bind to the affinity column was investigated. As shown in Figure 14, affinity chromatography of the dialyzed acetone precipitate prepared from Cohn fraction IV-l separated ceramide trihexosidase, Form A activity, into two protein peaks. These peaks were designated by their order of elution as A-1 and A-2. Ceramide trihexosidase, Form A-Z, binds to the column equally well in the presence and 121 .aficuflooa mo :ofiufipwm usogpflz xcmmhmoum50hso mm: oumuflmwoohm ocouoom wonxamflp one Amy .cwnuHoHH mo HE\wE w pocfimucou OmHm Hommsn mcflszo one .xcmmpmoumEOqu xwflcwmwm on poHHm H50: m.o How woumLSUCH mew oumuflmfioohm ocououm wouxamflw may ou wowwm mm: mHE\wE mv :Hnufiooq AHp coca .Hocmuzn wm wcwafimucoo .v.m :m .Howwsn mm: 2 Hoo.o umcfimwm woaxame mm: H->H cofluumww snow mo Em oom Eonm woummoha mumpflmfioohm oQOHoom use < Egon .omwwflmoxozflhh owHEmhou mEmmHm mo >5mmgmo~m50wcu xuflcflww< .wa ohsmfim 122 2,5 Ema—rm 8. .Q. 8 8...? 8.8 o. 8.0! _ 8.0a 1 N51 MM . m. 8 g. u ,I \ AA 4‘; l 2 l 8 745. g S3. 609 mpazfilmpflH 2-19 salowouON 2 Hxé q. WAN/9620mm baud-)0 salounuou c5 0 “- I .0. L 3 l CON 1 8 L 9‘; § > 00¢ 9 123 absence of lecithin, whereas Form A—l is retarded but not adsorbed to the column in the absence of lecithin. This property was selectively used for complete separation of the A-l form from other proteins in the preparation. The purity of enzyme A-l was 31,513-fold and that of enzyme A-2 was 35,600-fold greater than that of the initial extract. This fold-purification varied by as much as 4,000 depending upon the degree of contamination in the starting material. No interconversion of Form A-1 and A-2 was observed upon rechromatography of each fraction. The percentage of the total activity associated with each peak was consistent for a given preparation but not within different preparations. Affinity_Chromatography of Ceramide Trihexosidase, Form B: Affinity chromatography of the dialyzed acetone precipitate prepared from Cohn fraction I, shown in Figure 15, separated ceramide trihexosidase, Form B activity, into three protein peaks, designated by their order of elution as B-l, B-2 and B-3. If the individual peaks were allowed to stand in solution for 8-12 hours, rechromatography showed that peak B-l was partially converted to peak B-2, and peak B-2 was partially converted to peak B-3. There was no detectable conversion of peak B-3 to either B-l or B-2. Cellulose acetate electrophoresis of the pooled protein peaks, shown in Figure 16, separated these ceramide trihexo- sidases into five protein bands which could be stained for both activity and glyc0protein. Isoelectric focusing of the 124 .HE ow mo oESHo> Hmuou w :H :ESHoo poumHnHHflswo-on exp 0H onHQmm mmz oHQEmm onH .Hocmusn wm mcflnflmpcoo .N.n mm .Hommsn mumsmmocm E:H@0m z Ho.o Hmchmm womxamflp mm: H :oHHomHm czou mo Em oom Eouw popmmohm oumuflmfloopm occuoom och m Show .ommpfimoxozHHH ovHEmHou mammam mo xgmmgmoumEOHnu quchm< .mH oHsmHm 125 (--x-) ugaIOJd 6w K2 C? V, N — O O O' O O' I T V.) cm + - + ’<;:_-';r C.“ “Ll—ax? k EM: 7" a O Q >'< 1 Mk 1 l l O O O O O O O O oEoH usocufiz qu>Huom oflumexwam How onmmmm paw mesa uHuHmHmHHom m mo va may nufiz vowsHo opoz aHE m.oV mcofipomhm .oH pm muse; me How pcoHpmHm omOHUSm o>fiuhommsm m :H comsoow who: .H cofiuompm czou Eopm wouwmohm mumpflmwooum ocouoom wouxamfiw ocu wo HanahmoumEOHao xwficflmwm anw wocwmuno .mxmom :HoHOHm oze m Show .mommwflmoxosHHH o©HEmHou «Emmfim mo mcflm3oom oHHuooHoomH .NH opsmHm 130 (—).oszd 9 (IJQOVN ONN OON ow. mum: . ......COOO‘S O O O .0 O O O. O O H.m O -——-.- .3822 558... .9... ow. o o o o. o o o o on H 0 am 0‘. Hmm pH 00. A. O 0 mm . 8 8 8 H. H H-m \rlj O O a) V 8 (0414/me 2—19 sauowouoN 131 .qu>Hpom ommwfimOHomHmm -8 oflwwoomm-coc How paw .m @cm < mEHom .>HH>Huom ommmfimoxonflhu owHEmHoo How eoxmmmm who: :oHHumHH HE H Home 80 HH5 H.0v mposuHH< .H.m ma .HQHH:H mm: 2 Hoo.o EHHz woumpnflfiflsdo cesHoo quchmm oz» ow onaamm mm: oumuHmHoon o:ouoom pmeHme one ommpHmOXoLHHH owHEmHou xocwfix amen: mo xcmmhmoumEouzu xuflcflwm< .wH ohsmHm 132 2:: Ematm “ "u r I I I I I K K ‘1 I ‘4 \I II ‘1 <2 ‘3 (-*-)U!9101d 5w 9; O . 8 . ..O IpV‘T I WOOTX 8 C? l 1 Q 8 l 0 r0 §L O (\l 9v) O'QHd WWWH 9am» senuouoN 0 v- 0 O N 60) wpazfilmpm e19 saloumw O 0 r0 133 Figure 19. Comparison of Kidney and Plasma Ceramide Trihexosidases, Form A, by Cellulose Acetate ElectrOphoresis The ceramide trihexosidases obtained from the affinity column were concentrated against polyethylene glycol 6000. Protein (8 ug) was applied to the strips which were stained for activity. Electrophoresis was carried out at room temperature. The strips are (1) plasma Avl, (2) plasma A-2, and (3) kidney. The strips were redrawn for purposes of photography. 134 4.5- 'm r ‘1‘.’ 135 barium acetate precipitation, and diaflo (183). In each case the proteins either were not precipitated or were inactivated. It was found that enzymatic activity could be precipitated from Cohn fraction IV-l by 100% saturation with ammonium sulfate, but the enzymes obtained by this method were unstable, having half-lives of approximately 12 hours. The ceramide trihexosidases, Form A, appeared to bind to Sephadex G-75 and a 2,000-fold purification was obtained by chromatographing Cohn fraction IV-l on this gel. The enzymes were excluded in the void volume when chromatographed on Sephadex G-50. A combination of these two steps resulted in an impure enzyme preparation which was associated with lipoprotein, as judged by cellulose acetate electrophoresis of the protein followed by comparative staining for activity, protein and lipoprotein. Ceramide trihexosidase, Form A, was separated into two enzymatically active fractions by elution of Cohn fraction IV-l from DEAE-cellulose using a sodium chloride gradient (0.05-0.8 M). This procedure resulted in a 5% purification accompanied by loss of about 50% of the enzymatic activity. CHARACTERIZATION OF THE CBRAMIDE TRIHEXOSIDASES, FORM A Substrate Specificity The purification of a-galactosidases from Cohn fraction IV-l was monitored with GL-3, 4-methylumbe11iferyl-o- galactoside and p-nitrophenyl-a-galactoside as substrates. 136 The specific activities in the crude Cohn fraction and the dialyzed acetone precipitate, as determined with the various substrates, are shown in Table 12. During the purification two discrepancies indicated that the glycolipid and artificial substrates were not measuring the same enzymatic activity. There was a partial loss of the a-galactosidase activity detected with the artificial substrates, whereas there was no decrease in the enzymatic activity measured with GL-3. Furthermore, the fold-purification measured with the artificial substrates could not be correlated with that obtained using GL-3 as substrate, even after taking into account the loss of non- specific a-galactosidase activity. Figure 20 shows the elution profile of the dialyzed au:etone precipitate from the affinity column. Two fractions (If ceramide trihexosidase were obtained, neither of which hiid detectable activity toward the artificial substrates. Ir1 addition, a single fraction was obtained that hydrolyzed bCIth 4-methylumbelliferyl-a-ga1actoside and p-nitrophenyl- o“'galactoside, but had no detectable activity toward GL-3. Ijllxs affinity chromatography resulted in complete separation (”fr the GL—3-c1eaving a-galactosidases from the enzymes hYdrolyzing the artificial substrates. .lgpvestigation of the Enzymes used in Sequencing GL-3 In view of the specificity for lipid substrates shown ID)’ the ceramide trihexosidases, it was of interest to determine whether a-galactosidases obtained from ficin, 137 Table 12. Comparison of Enzymatic Activity using Natural and Artificial Substrates The Cohn fraction and dialyzed acetone precipitate were assayed for enzymatic activity as described in Materials and Methods. In this case sodium chloride and sodium taurocholate were added to assay ceramide trihexosidase activity in the dialyzed acetone precipitate. Specific Activity nmoles (mg protein)'1 (hr)-1 59ubstrate Cohn frac. IV-l Dialyzed Acetone Ppt. CTH 0.07 138 <1-4-MU 0.12 92 cx-pNPG 0.10 76 x 138 .moumpumnzm cfimfla mam Hmfiofimwupw :uon maflm: qu>Hpom oHumEchm How woxmmmm who: cowpumpm HE H :omo mo flHE H.0v muoscfla< .H->H :oHpome czou mo Em omN Eouw woummmhm opmufimfiooum ocouoom mowxamflv esp mo xnmmHmoumEOHno zuHcwa< < Show .mommemoxoLHHH opHEmHou mEmmHm mo kuHonHooam opmHHmQSm .oN opsmHm 139 +) UlelOJd 6w A 8:» s a. a a as 8 ¢--=: K8 8 - . ' 49 8 8 i (H OOI-xuowl' 8 8 8 .9 T ({J T (1) r (Y:- " ‘5 O — N (——o—) 10 [p32A|OJpAH pde-xnsaloguu r <5 c5 <5 0 5 o o N LO 0 <1' (I) N (0 <1" K) K) N — (+)JH/PGZAIOJPAH €519 SSIOUJU Effluent (ml) 140 reportedly hydrolyzing GL-Zb and GL-3 as well as non—specific o-galactoside substrates (184), might contain an enzyme specific for hydrolysis of GL-3. Affinity chromatography of ficin, shown in Figure 21, revealed three primary peaks of a-galactosidase activity. One of these hydrolyzed both lipid and non-specific a-galactoside substrates, one hydrolyzed only the artificial substrate, and one was specific for hydrolysis of GL-3. Electrophoretic Properties ElectrOphoretic investigation of the ceramide trihexo- sidases, Form A, was undertaken to obtain information regarding the purity of these enzymes as well as to delineate any differences between the two forms. Isoelectric Focusing: Isoelectric focusing (Figure 22) of the ceramide trihexosidases, Form A, showed that both forms of the enzyme have a pI of 3.0, thereby indicating that both proteins have the same electrical charge. In both cases all of the detectable protein was associated with ceramide trihexosidase activity. The specific activity of both enzymes before and after isoelectric focusing was constant within experimental error and the same as that obtained following affinity chromatography. Polyacrylamide Gel ElectrOphoresis: The proteins obtained by affinity chromatography were electrophoresed on 1% SDS, 9% polyacrylamide gels as described in Materials and 141 .poschcoo mm: coausHo wcm Mommas may o» @8888 mm: QCH-x couHHH wH.o soap .oHnmuoopow mm: :Houopm on Haas: Mommas :HHB wousao mmz :EsHoo 8:9 .Homwsn oEmm may cpflz woumHnHHfisco coon mm: :UHLB :Esaoo xuflcfimmm ozu ou meHmmm paw .m.w :m .Howm39 oumuoom Esflpom 2 mo.o :H po>H0mme mmz Ame oav :Hofim :Hoflm mo xsmmuwoumEowgu kacwa< .HN ohsmHm 142 (-*-)ugalmd 6w 51'. V0. N. 5 O C? C? I 8 —8 *8 ‘9 -8 *8 A% C r—. —9 8 X ¢===-—;:foé<:g 8 —Q .2. I .5 s .- 9. — JL| /pazA|01pKH 910.415an selowouoN (4799'de m Sde-Do (0)17“st l0 919 Effluent (ml) 143 .mouxflonmEm HoHHHmo onu mo Hm>oth usonuwz xufi>fluom oflpoaano Mom poxmmmm ohoz “HE NV mnofiuoopw one .OH no mnson NA 90% uconon omOHosm o>HuHomm3m o cH womsoom oHoz :Esfioo HoHcHHoa map Echo eocHaHno Hmo N-< one Hohnnm one .oESHo> cEDHou u u> paw mcHoHOHQ onu mo oESHo> nofiusao u o> .cohuxoa osfim mo oEDHo> :oHusHo o> ohonz mo> - u>v\mo> - o>v u >mn oHDEHom onu Eouw nonmasuamu mo: >m¥ onp .mao mm x my :ESHoo Em-< How on o no momownmoxonwhu owfleohoo onu mo xnmohmoumEOHnu new new nun .mucowmou mnHosvon HxhvxanSm paw mom mo ounomohm on» :H mEo mm x mv cESHoo Em-< Hoo on o no momovaOXonHHp opHEmHoo on» mo xnmonwouoEownu Amy mam m0 151 .m wonuoz kn poEHOMHoQ oHoz mxommo wnm .m-no moHHoancs mam m-nQHUVHH mo oHSHXHE o an3 H30 onHHmo oaoz mnoHHmnsocH Hmo N-< anon new A Hmcflm o :H oumHonooHSmu Esfiwom mo mcoHHmHunoocoo mcflmmohonfl cam .e.m :m .Howmsn ouonmmonm-ouohuflu .opflhoano Sawnom 2 mH.o .omomeOXoanu owHEoHoo m: m .ouoaonu Ezflvom we H :H poNHHanaom m-no oHoE: N.o wonflmucoo mnoflpmnsunfi one H-< Show .ommvflmoxonwnh opHEmHou no oumHonUOHsmH EsHpom we uoowwm onu wcflzonm uon npsmrno>oo3onfin .Amo3ocHn .H00 00 onsta 161 725.032 4 m m H H 1¢N 162 concentrations of sodium taurocholate decreased this sigmoidality but did not affect the maximal velocity. At optimal concentrations of sodium taurocholate (0.04 M for Form A-2 and 0.03 M for Form A-1) and 0.15 M sodium chloride the lag phase which occurs at low substrate concentrations was completely eliminated. Under these conditions, half- 4 maximal velocity occurred at 4.5 x 10' M GL-3 for Form A-1 and at 5.0 x 10-4 M GL-3 for Form A-Z. The Km's for j ceramide trihexosidases, Form A-1 and A—2, determined from the double reciprocal plot of velocity vs. substrate 4 concentration, were 2.1 x 10' M and 2.2 x 10'4 M, respectively. Effect of Engyme Concentration on Hydrolysis of GL-3: The effect of ceramide trihexosidase, Form A-l, on the hydrolysis of GL-3 was compared in the presence and absence of both activators and inhibitors. The results of this experiment, shown in Figure 27, demonstrated that the enzymatic activity at a particular substrate concentration was dependent on both the enzyme concentration and the presence of activators or inhibitors. At a substrate 4 concentration of 4 x 10' M or greater and in the absence of activators or inhibitors, the enzyme becomes inactive 7 M, whereas with 4 when its concentration exceeds 1.68 x 10' substrate concentrations of less than 4 x 10' M, the enzyme becomes inactive at a lower enzyme concentration. 163 Figure 27. Effect of Enzyme Concentration on the Hydrolysis of GL-3 The assays were carried out in volumes ranging from 0.2 ml to 1.0 ml, so that each incubation in a series contained a constant concentration of GL-3 as shown in the figure plus 10,000 cpm of [14C]GL-3, 8 ug of ceramide trihexosidase, Form A-l, 1 mg of sodium cholate, 0.15 M sodium chloride, and sodium citrate-phosphate buffer, pH 5.4. Incubations with detergent contained 0.03 M sodium taurocholate; those with inhibitor contained 0.2 umole of GL-Zb. The concentrations of reagents were kept constant by serial dilution of concentrated solutions. ysis cpm GL-Zo Liberaled(8}19 ).'(hrl-| 164 I000 I 800* 600~ 200- I400 IZOOH IOOOr 200- CONTROL/ +IM-IIBI'I'OR/ «momma 4xlo“‘M GL-3 H_¢1, ‘04} l I I l200- IOOO- 400- 200- l l l I 2 3 [Elxlo’M .pp 165 The addition of sodium taurocholate and salt to the system results in loss of activity at higher enzyme concentrations. Although the point at which inactivation begins is not altered with a substrate concentration of 4 x 10’4 M, the inactivation occurs at a slower rate with increasing enzyme concentration. As shown in Figure 28, the enzyme is activated at low substrate concentrations and inhibited at high substrate concentrations in the presence of an inhibitor (GL-2b) when sodium taurocholate is omitted from the incubation. i From this data it was postulated that linear kinetics could be obtained in the presence of sodium taurocholate and sodium chloride in the range of substrate concentrations being studied, if the enzyme concentration was 1.68 x 10'7 M or less. Inhibition Studies: The various inhibitors of each of the ceramide trihexosidases were compared under a single enzyme concentration of 1.68 x 10'7 M. All incubations, buffered at pH 5.4, contained the optimal concentrations of detergent and salt. Under these conditions, plasma ceramide trihexosidase, Form A-l, was competitively inhibited by trisaccharide [galactosyl(o1+4)galactosyl(81+4)- glucose] and GL-Zb, as shown in Figure 29. As shown in Figure 30, ceramide trihexosidase, Form A-Z, was inhibited by the products of the reaction, galactose and GL-Za. In addition it was competitively 166 .H-n-0 z 0-0H x 0 000 ”H-0-0 z 0-0H x N ”H-.-0 2 HTOH x H ”H-o-v :oHquwm on unm-nu mo mnonoHunoonoo wcHonHom onu wocHoucoo meHumnzocH one .ohsuxHE coHumnsonH on» Scum ouoHonUOHsmu Eusom can owHHoHno EsHvom Ho :onmHEo onu anz m nonpoz wnHm: poHSmmoE mm: >HH>Hpom oHumsxmcm m-Ho Ho mHmHHonenz 00H 00 0N-Hu Ho Hoooom 809 .0N onsta 167 2.10. 352 m o v \fi _ H H H H CON 0 0 q- C C to wnamqn 02-19[o,,] wdo O O (n 000. ICON. 168 .H-o-0 :5 0.0 0:0 H-<-0 :5 N.0 ohm mcoHpmuunoocoo HOHHancH one .VOH x H-Hpn\woumnonHH om-HoHCvHH EmoH mo wommonmxo mH >\H .m wonuoz mch: wonHEHouow mmz >0H>Huom oHuoEHNnm H-< Show .ommemoxonHHH owHthoC no owHHwnooomHHH mo uoowwm xpoanHnnH onu mnHzonm uon nu3m-po>mo3ocHn .H\H .m wonuoz mnHm: wonHEHopow mo: po>Huom oHumEchm H-< Egon .omovaoxonHHH ovHEmHoC no nN-no Ho Hoowwm xHoanHnnH onu mnHzonm uon nhsm-Ho>mo3ocHn .H00 00 onsmH: 025.032 C. m C to N C N- Hu- H H H H H H .20: H: 0.0 mo Mo 6 o , .m 171 O C_ 9 ON Cm 172 .H- -0 z: 0.0 0:0 H-0-0 :0 0.0 H-0-0 z: H.0 on mcoHumenoocoo HoanHnnH one .0-OH x H-th\poumhonHH 0N-HCHC0HH EmoH mo commopaxo mH >\H .m wonuoz mch: ponHEHouow mm: >HH>Huoo oHuoechm N-< Show .ommemoxonHHH owHEoHoC co omouomHoo mo uoommm xuoananH on» mcHzonm uon Hhsm-Ho>oo3o:HH .H<0 om oHsmHm 173 ...25 .0300 N 174 .< upon How mo oEom onu mH wnomoH one N-< Show .omomeoxonHee owHEoHoC no mN-Ho Ho poommm xHoananH onu mcHzonm HOHN nu:m-po>mo3o:Hn .H00 00 ousmH: 175 0.2:. ..Hm-Ho H o. m o N o N- H0- H H H H H H H 255 0.0 no No a o H H 0 1 m C_ 0. CN mN 176 .< puma How mm oEmm onp mH pnoon one N-< Show .omomeoxonHHe omHEmHoC no owHHmnoommHHe Ho uoommm xHoananH onu mnHzonm uon nu:m-uo>oo3onHH .H00 00 0::0H0 177 7.250358 C. m w v C Hw- @- H H 0 H H J 255 .0 0.0 N0 0 o S m . 1N3 000 . 10. 0m 0 . . . . . ..m. . >\_ JON . -mN 10m 0 000 178 .< whom How mo oEom onu mH wcomoH one N-< Show .ommemoxonHHe owHEoHoC no nN-no Ho poommm xpoanHnnH onu mnHzonm uon Hesm-Ho>oo3o:Hn .H00 00 00:0HH 179 180 .< Hung Hom mm oEom onu mH wcoon one N-< Show .ommemoxonHHe owHEmHoC no HouHmonH-oms mo poomwm xhoananH onu mnHzonm HOHm HHDm-Ho>mo3onHH .Hm0 0m 0530 181 r! I'll! hi qlu C. m H H-EEL MACH H H 2:. H: 182 inhibited by GL-2b, trisaccharide and inositol. Under the conditions of this experiment neither form of the enzyme was inhibited by either p-nitrophenyl-a-galactoside or 4- methylumbelliferyl-a-galactoside as recently reported by Ho (123). The kinetic parameters for the ceramide trihexosidases, as determined by computer analysis, are summarized in Table 16. Classification by Gatt's Kinetic System: Bovine serum albumin and lecithin were used to form a pseudo- micellar system for hydrolysis of trisaccharide in the manner described in Materials and Methods (Method 5). The double reciprocal plots of velocity vs. trisaccharide concentration are shown in Figure 31. Ceramide trihexosidases, Form A-l has a non-symmetrical sigmoidal velocity v3. substrate saturation curve and according to Gatt's classification scheme is a Type IV sphingolipid hydrolase. This is the case in which the enzyme utilizes micelles but not monomers (124). Form A-Z has the classical Michaelis-Menton type V/S curve, under the conditions of this eXperiment, and is classified as a Type I enzyme. This type of curve is obtained when there is no monomer-micelle transition of the substrate (124). 183 Table 16. Summary of Kinetic Parameters Kinetic analyses were made using the ModHill computer program. Ki's were calculated from the intercept of the slepe v3. intercept plots for the various inhibitors. In the cases where this plot was not a straight line the Ki was determined using the tangent to the curved line. Ceramide Trihexosidase, Form A—l v = 4.5 x 10'4 M max K = 2.3 x 10‘4 M m Ki (GL-Zb) = 3.0 x 10'4 M Ki (Trisaccharide) = 8.3 x 10.4 M Ceramide Trihexosidase, Form A-2 _ -4 vmax - 5.0 x 10 M _ -4 Km - 2.5 x 10 M Ki (Galactose) = 7.0 x 10'4 M Ki (GL-Za) = 5.0 x 10'4 M Ki (myo-Inositol) = 4.3 x 10-4 M Ki (Trisaccharide) = 2.3 x 10'4 M Ki (GL-Zb) = 1.2 x 10‘3 M 184 m H- . OH x Hen N\woumnonHH omouomHmm mo moHoecH mm pommopmxo mH >\H .m ponuoz Nch: wocHEHouow mm: euH>Huuo oHuoexmnm HHH>HHU< ommemoxonHHe opHEmHou no :oHpoHuaoonou owHHmnooomHHe wnHmmoHonH mo uoommm one .Hm onsmHH 185 CV CC. 186 a-GALACTOSIDASES OF WHOLE PLASMA Affinity Chromatggraphy of Normal Plasma Whole plasma was investigated by affinity chromatography to determine whether the ceramide trihexosidases could be studied without fractionation of plasma. As shown in Figure 32A all of the ceramide trihexosidases obtained by Cohn ”0“} fractionation of plasma were adsorbed to the affinity column and eluted in nearly the same fractions as the partially purified enzymes. However it should be noted that in whole plasma the ceramide trihexosidases, Form A, are present in nearly equal quantities; whereas Cohn fraction IV-l contains more Form A-2 than Form A-l. The fold-purification for the ceramide trihexosidases, Forms A and B, determined by comparison of the specific activity obtained after affinity chromatography with that obtained for whole plasma are 48,989 and 12,676, respectively. Polyacrylamide electrOphoresis indicated that Form A-l was about 90% pure and Form A-2 was about 50% pure. In addition to the ceramide trihexosidases six non- specific a-galactosidases were obtained by affinity chromatography of whole plasma. The four a-galactosidases hydrolyzing p-nitrOphenyl-a-galactoside at pH 3.0 were designated PN-A and were numbered in the order in which they were eluted; the two a-galactosidases, catalytically active at pH 6.0 were designated PN-B-l and PN-B-Z. 187 .mcoHumHunoonoo nHoHOHQ mammHa :H mnoHumHHm> Host>HpaH How owme moz :oHnooHHoo oz .oEmmHm me He ooH mo oEsHo> unoumnoo m on vouoohhoo noon o>on wumw one .mEmon xnnmm mDONHNHEon mo enemymoumEOHno HuHchw< HCH cam memon xenon msowxmoponon Ho xnmmamoumEoHno AHHQHHH< HmH HoEmmHm HoEHo: Ho xnmoumonmEoeno annHwH< H<0 .0.0 m: .00HH00 mm: 2 H00.0 :0Hz 000000HHH000 :esHoo 00H:HHH0 0:0 0000000 wonoHoupom cam wHom oHHuHu z H.0 nqu 0.m mm on wopmsnwm mm: oEmmHm oHonz «Emon oHonz Ho xnmmHNOHmEOHnC HHHCHHH< .Nm onsmHm I 3 S 3 9 (-'-)9Hd l 8 (+I21Hd 188 (He) upload bu: WWW sand-)0 sermon ééééé III/902001944 2-‘19 80mm (402m I 3 W9 3 no 1 8 A aoao4osoeomaosooo IO Effluent (ml) 189 Affinity Chromatography of Fabry Plasma In an attempt to determine whether there was an actual deficiency of ceramide trihexosidase in Fabry's disease or rather a genetic alteration resulting in a catalytically inactive protein, the multiple forms of ceramide trihexo- sidase in both heterozygous and hemizygous Fabry plasma were investigated. Affinity chromatography, shown in Figure 32B and C revealed the presence of both of the ceramide trihexosidases, Form A, which occur in normal plasma. The total enzymatic activity of both of the A forms obtained from hemizygous Fabry plasma was 1-2% that of normal; whereas Form A-l, obtained from heterozygous Fabry plasma, had 50% of normal activity and Form A-2 had from 10-30% of normal activity. In addition to the a-galactosidases Fabry plasma from heterozygotes and hemizygotes contained catalytically inactive proteins which were not observed in normal plasma. These proteins were associated with the A-l form of ceramide trihexosidase, and cellulose acetate electro- phoresis of this fraction from the affinity column (Fractions 39—50 in Figure 33) indicated that they were glycoproteins of similar electrophoretic mobilities. In contrast to the Form A enzymes there was a total absence, in both hemizygous and heterozygous Fabry plasma, of all the proteins associated with the enzymatic activity of ceramide trihexosidase, Form B. This finding suggests 190 Figure 33. Comparison of Normal and Fabry Ceramide Trihexosidase by Cellulose Acetate Electrophoresis The proteins obtained from the affinity column were concentrated against polyethylene glycol 6000. The cellulose acetate strips were electrophoresed at room temperature and stained for glycoprotein. The strips are (1) normal plasma, A-l; (2) heterozygous Fabry plasma, A-l; (3) hemizygous Fabry plasma, A-l; (4) normal plasma, A-2; (5) heterozygous Fabry plasma, A-2; and (6) hemizygous plasma, A-2. Lmide lumn were 'Ihe celluhn Iperature :1) normal Iasma, A'R nizngUS 1591 w ,_ W M .M 6 mi “*0 in.“ man-“’0‘" ‘IIIIIIIII lksnai _ N N N N 192 that the ceramide trihexosidases, Form B, were converted to the catalytically inactive proteins which accumulate in Fabry plasma. In addition to the alteration of the proteins having ceramide trihexosidase activity, the activity of the non- specific o-galactosidase, PN-A-Z was depressed in both hemizygous and heterozygous Fabry plasma, whereas the E_‘ activity of PN-A-3 was elevated. A consistent pattern of protein and enzymatic activity, summarized in Table 17, was obtained from the plasma of i eight different Fabry's, although the ratio of the [0 individual proteins and their activity showed some variations. The finding that only one of the non-specific a- galactosidases in Fabry plasma had depressed activity is not compatible with the success of artificial substrates in diagnosing Fabry's disease. Therefore the a-galactosidase activity in whole plasma, as summarized in Table 18, was compared before and after affinity chromatography. Prior to affinity chromatography the non-specific a galactosidase activity measured at pH 3.0 was depressed in Fabry plasma. Hemizygous plasma had approximately 20% of normal activity, while the activity in heterozygous Fabry plasma was from 70-90% that of normal. Following affinity chromatography, the combined activity of the PN-A a-galactosidases, obtained from both hemizygous and heterozygous Fabry plasma, was about 98% of that observed 193 eH.o eH.o nH.o mH.o 0H.o mH.o 0H.o 0H.o mH.o mH.o «H.0 NH.o 0H.o mH.o NH.o mH.o mH.o NH.o OH.o 0H.o mH.o «H.0 0H.o MH.o 0H.o mH.o mH.o Nm.o mm.o No.0 Nw.o Hm.o Nm.o + + + + + + mo.o Nm.o mH.o mN.o v~.o 0H.o eH.o NH.o NH.o Ho.ov Ho.ov Ho.ov eH.o eH.o 0H.o Ho.ov Ho.ov Ho.ov 0H.o nH.o mH.o Ho.ov Ho.ov Ho.ov 0H.o NH.o 0H.o Ho.ov Ho.ov Ho.ov eH.o mH.o mH.o Ho.ov Ho.ov Ho.ov mH.o MH.o NH.o Ho.cv Ho.ov Ho.ov eH.o NH.o NH.o Ho.ov Ho.ov Ho.ov 0H.o mH.o NH.o Ho.ov Ho.ov Ho.ov mH.o mH.o mHHo Ho.ov Ho.ov Ho.ov mum-2m Ham-2m vichH onu pom wonHmuno kuH>Huo< new nHonoum onu Ho Humeesm xnmmhmouoEOHnu euHaHmm< wcH3oHHom momomeopomHmo-a .eH oHnoe 194 00H>Huo< Hmuoe 0.mH 0.mH 0.mH 0.mH 0.mm m.mH 0.m~ 0.mN 0.m~ 0.m~ 0.m~ + + + + + + + + + + + m.00 m.0m 0.00 0.0H m.m0 0.00 0.0m0 0.000 0.000 0.000 0.0N0 HoHuc0 mHmEHoz N.N0 0.0m 0.00 0.00 m.mH 0.00 0.Hv 0.Hv 0.Hv H.0mH 0.00m .H.u n.00 0.Nm 0.0m 0.00 0.0H 0.00 0.Hv 0.Hv 0.Hv 0.mmH 0.00m .H.: 0.00 0.0m 0.0m H.N0 N.0H m.00 0.Hv 0.Hv 0.Hv 0.0NH 0.HNm .0.m monomeNOHouom N.00 0.Nm 0.m0 n.00 m.0H ~.00 0.Hv 0.Hv 0.Hv 0.0 0.0H .H.m 0.0m H.00 0.00 N.00 0.0 m.00 0.Hv 0.Hv 0.Hv 0.0 0.mH .0.2 0.00 N.0m 0.0m 0.00 N.0H H.N0 0.Hv 0.Hv 0.Hv 0.0 0.0H .H.C 0.00 0.0m N.00 0.00 m.NH 0.00 0.Hv 0.Hv 0.Hv 0.0 0.0H .n.m 0.n0 0.0m 0.00 0.00 m.0H 0.00 0.Hv 0.Hv 0.Hv 0.0 0.mH .H.om 0.00 0.00 H.m0 0.00 0.HH 0.00 0.Hv 0.Hv 0.Hv 0.0 0.HH .H.Hm ~-m-zm H-0-20 0-<-zm m-<-zm N-<-zm H-<-zm m-m N-m H-0 N-< H-< monomquEon Hn\0ouxHouwxm umzmwa moHoa: Hn\0omNH0000: m-Hu moHoE: 195 .0erao mm 0000 um 00.H-m.0 0n 0lom> moquHHmsv Hnmmnmoumsonno HuHcmem mnHonHom .0.0 mm 00 00-0 0n 0cm 0.m :0 am wemmHa oHon3 mchs on-m 0n 0oHHm> moumoHHmsn .0ommno>m ouos oumnumnSm mm owHwonomHmm-e-H0aonmouuHc-m mnHNHHHu: m0mmmm oumoHHmsn xnamnmoumsoenu 0anHmm< Houm< 0cm onowom 0HH>HHU< omm0HmouomHmo-d «EmmHm onu mo nomHHmmEou .wH oHnme 196 000000H 000000H 00000 H00H00 000000H 000000 00000 000000 H0Hu:0 0H0::0z 0.Hv 0.H00 0.00 0.00H 0.Hv 0.000 0.00 0.00H .H.m 0.Hv 0.000 0.00 0.00H 0.Hv 0.000 0.00 0.00H .H.o 0.Hv 0.000 0.00 0.00H 0.Hv 0.000 0.00 0.00H .0.0 monomxnohouon 0.Hv 0.00 0.00 0.00H 0.Hv 0.Hv 0.00 0.00 .0.2 0.Hv 0.0H 0.00 0.00H 0.Hv 0.Hv 0.00 0.00 .H.0 0.Hv 0.H0 0.00 H.00H 0.Hv 0.Hv 0.00 0.00 .H.o 0.Hv 0.00 0.00 0.00H 0.Hv 0.Hv 0.H0 0.00 .H.0m 0.Hv 0.0H 0.00 H.00H 0.Hv 0.Hv 0.00 0.00 .H.Hm 0.Hv 0.0H 0.00 0.00H 0.Hv 0.Hv H.00 0.00 .H.o mou0000HEon H-HHEdeHH Hann0 0000000000 00Hoe: 0.0 mm 0.0 00 0.0 :0 0.0 :mi 0.0 mm, 0.0 mm 0.0 mm- 0.0 mm :00 0020-0 000 0020-0 0nmmhmoumEonnC 0nmmnmoumsounu 00H:Hmm< 0:H20HH00 00H>H00< 00H:H00< 000000 00H>H00< 0000000 197 ivith normal plasma. These findings suggest that there is some type of inhibitor for the non-Specific a-galactosidases in Fabry plasma which is removed by affinity chromatography. Investigation of Digalactosylceramide:Galactosyl Hydrolase in Human Plasma As shown in Figure 34, Optimal hydrolysis of GL-Zb :_ occurred at pH 5.5. Since hydrolysis of GL-Zb by the A 2 and B forms of ceramide trihexosidase was negligible, the : proteins obtained by affinity chromatography of whole ; plasma were assayed to determine whether one of the non— 3 specific a-galactosidases coincided with an enzyme which would hydrolyze GL-Zb (digalactosylceramide). The results of affinity chromatography of normal and Fabry plasma, illustrated in Figure 35, showed that the a-galactosidase hydrolyzing GL-Zb in normal plasma is the same enzyme which shows depressed activity toward the artificial substrate, p-nitrophenyl-a-galactoside, in Fabry plasma. Electrophoretic Investigation of Digalactosylceramide: Galactosyl Hydrolase: The enzymatic activity coincident with hydrolysis of both p-nitrophenyl-a-galactoside and GL-Zb was investigated by isoelectric focusing. There were two primary protein peaks having pI's of 4.2 and 4.6, as shown in Figure 36. Both of these proteins were equally effective in cleaving either the artificial or natural substrates. 198 o.m :m .uommsn oumgmmoam-oumhpflo z H.o £003 wopmznmm mm: mm 0:0 .H->H :ofluumum anou mo c00usfiom m mqfim: cocfimpno mm: Ezeflumo :9 mo o>Hsu 0:0 00-00 00 000000000: 000 :0 00 00 000000 .00 000000 l 9 E) l l l I a e .9. a 2 ,Jw- ,yz- pazKImMH Caz-19 salauouou 200 .mEmmHm mo 05 OOH mo oESHo> 0:000:00 m 00 pouoohhoo 0003 0000 0:0 mammam xunmm msom0005o: mo xnmmhmoumEouco xuficdmm< 0mg «Emmam H0500: mo xcmmnwoumthno xuficwmm< Aamm~wo~05000u 000c000< .mm opsmflm 201 (—n—)ug9401d 5w 8 8 5 I I I osoh 0300003 xufi>00om oflumfixuco 000 0000000 @003 has NV mnofipumhm 000300 one .oH am 0: m0 000 pcoflvmhm amouuzm o>000omm50 m :0 0003000 who: 0nmmuw00050030 xpflcwwmm Eoum cocflmuno mcflopopm 0:5 ommHOwam 0000000000 0 owMEmpooaxmouumHmmfim mo mafimsoom ofihpoonOmH .om opsmflm 203 (—-)ooz 1o Hd 9 co co <1- N I l l I 8 210 A 1; . 20.. IO l l B 98 1 Q (-V—)L|/paZK|OJpK|—| ngd-po salowouoN 1 1 1 J 1 1 S: [\- L 1 00000 8 a 2 <2 «0 (...)u/pezKIOJp/(H qzrlg salowouoN 36 Fraction Number 204 Comparison of normal and Fabry ceramide digalactosidase activity by cellulose acetate electrophoresis, shown in Figure 37, revealed that there are two enzymatically active components in normal plasma, whereas only the enzyme of slower electr0phoretic mobility is present in Fabry plasma. URINARY CBRAMIDE TRIHEXOSIDASES Pen Crude urine was assayed for ceramide trihexosidase activity using galactose dehydrogenase (Method 1). It was found that the ceramide trihexosidases are excreted in the urine, with about three times as much activity associated with Form B as with Form A. As shown in Figure 38, these enzymes have the same binding capacity on the affinity column as the plasma enzymes and are eluted in nearly the same fractions. The concentration of the ceramide trihexo- sidases in urine was estimated to be 50 pg per liter for Form A and 170 pg per liter for Form B. INTERCONVERSION OF THE CERAMIDE TRIHEXOSIDASES Preliminary Neuraminidase Egperiments The effect of neuraminidase on the enzymatic activity of the ceramide trihexosidases was studied using the crude A forms obtained from Cohn fraction IV-l. Cohn fraction IV-l, having detectable enzymatic activity at only pH 5.4 (Form A) was incubated with commercially purified neuraminidase from CZostridium perfringens. The effect of neuraminidase on the pH optimum of ceramide trihexosidase, 205 Figure 37. Comparison of Normal and Fabry Digalactosyl- ceramide : Galactosyl Hydrolase Activity by Cellulose Acetate Electrophoresis The proteins obtained from affinity chromatography were concentrated against polyethylene glycol 6000 and electrophoresed at room temperature. The strips are (1) normal enzyme stained for glycoprotein, (2) normal enzyme stained for activity, and (3) hemizygous Fabry enzyme stained for activity. The strips were redrawn for purposes of photography. osyl- y by aphy and 11131 wn for 2()€5 fl‘. ; :1; 1’th W7?" q-‘am «It-W Ml”.' 1 207 .000030 mm: 2 0oo.o c003 0.m $0 00 000000000300 053000 00000000 000 00 0000000 000 0000 000000 2 0.0 0003 v.m :0 00 00003000 003 00003 00 000000 m 00 00000>0300 0030000 c< .500000 000000000000003 00000 30000: :000E< :0 mc0m3 0000-m0 000000000000 003 0:00: 0:00: 000000000000 00 00000m000500zu 0000000< .wm 003M00 208 (+101on am 60 4O 20 0. IS #012 3008 -004 IOO «~04--- 8 E E 83 t LIJ T . . J r_. < “oat. 8 0 S2 >'< T g <)r=:;';';,__:~ 8 8 0 _________.___.x-—\ 0 c3 6: a v m N - Ju/ pazKIOJpKH who SSIO‘UOUDN Eangl 1 IE aEHdl 8 8 8 8 0 _ _ Ju/ pazfilonKH 9‘19 salowouoN (+)ZZHd 609179 Hd 209 Form A, is shown in Figure 39. Following neuraminidase treatment there was substantial activity at pH 7.2 (Form B), but at pH 5.4 the activity had disappeared completely, within the limits of the method of assay. The control had no detectable activity at pH 7.2 although 30% of the ceramide trihexosidase activity at pH 5.4 was lost during the incubation. This loss of activity is explained by the instability of Form A, which had a half-life of 5 hours under the conditions of the experiment. Incubation of whole plasma with neuraminidase enabled a study of the time course of the reaction, which shows a correlation of sialic acid release with the change in enzymatic activity at both pH Optima, as shown in Figure 40. There was no detectable release of sialic acid in the controls, although there appeared to be a similar, though less marked, conversion of the ceramide trihexosidases, Form A to Form B during the first hour of the incubation. Thereafter both enzymes progressively lost activity in the control samples. A second series of controls contained 2-4 -3 x 10 M N-acetylneuraminic acid, which neither inhibited enzymatic activity at pH 5.4 nor enhanced activity at pH 7.2. Neuraminidase Treatment of Purified Ceramide Trihexosidase, Form A-l Electrophoretically pure ceramide trihexosidase, Form A-l, was treated with neuraminidase purified by affinity chromatography. Ceramide trihexosidase (300 ug) was :‘:_... 210 .0000002 000 000000002 00 000000000 00 030 0000000 00000003000 0-0-0 0000000 000 0-0-0 0000000 0000000000300 0003000 00 0000000000 000 .0000000000300 00002000000 00000000000 00000000 000000000000 0003 0000000 0003 0->0 00000000 0000 0000 00>0000 .< 0000 .00000000X00000 00000000 < 0000 .00000000x00000 00000000 000 00 0300000 :0 000 00 0000000000302 00 000000 .00 003M00 211 20 *— 7.8 7.0 6.2 pH 5.4 4.6 I l l 0 n 9 l0 0 Squ/lw/samwu All/\ILDV DILVWAZNQ 212 Figure 40 (A). Correlation of Sialic Acid Release with Changes in Enzymatic Activity Time course showing the correlation of sialic acid released with decreased enzymatic activity at pH 5.4 when normal human plasma is treated with neuraminidase. Enzymatic activity at pH 5.4 in the presence of neuraminidase (-o-); the loss of enzymatic activity in the absence of neuraminidase (--o--); and the level of sialic acid in the presence (-I-) and absence (--I—-) of neuraminidase. 213 O. 4 _E\00_oEl 0mm._._>:.U< U_h<<<>NZw 3 2 TIME (hrs) 214 Figure 40 (B). Correlation of Sialic Acid Release with Changes in Enzymatic Activity Enzymatic activity at pH 7.2 in the presence (-o-) and absence (--o--) of neuraminidase. 215 30— 000.01.800.08: _>._._>:. U< U_._.<<<>NZm 3 2 TIME (hrs) 216 incubated with 50 units of neuraminidase for 4 hours. The time course of the reaction was followed by cellulose acetate electr0phoresis and isoelectric focusing. As shown in Figure 41, one hour of neuraminidase treatment altered the activity of ceramide trihexosidase, Form A—l, by forming 8 proteins of lower electrOphoretic mobility. Isoelectric focusing of the ceramide trihexo- sidases after 2 hours incubation with neuraminidase, shown in Figure 42, gave a complex pattern of 14 enzymatically active proteins. Seven of these proteins were active at pH 5.4 (Form A) and the other proteins were active at pH 7.2 (Form B). One of the proteins active at pH 5.4 had a pI of 3.0 and probably represents unchanged ceramide trihexosidase, Form A-l. One of the proteins enzymatically active at pH 7.2 had a pI of 8.4 which is the same as the pI of ceramide trihexosidase, Form B-V. After a 4 hour incubation with neuraminidase (Figure 41) ceramide trihexo- sidase, Form A-l, was not detectable but an enzyme having the same electrOphoretic mobility as ceramide trihexosidase, Form B-V, was present in addition to two proteins of inter- mediate electrophoretic mobility. In the control, incubated 4 hours without neuraminidase, the majority of the protein had the same electrophoretic mobility as ceramide trihexosidase, Form A-l, although two proteins of slower electrophoretic mobility were found. These results suggest that ceramide trihexosidase, Form A-l, .lj ‘ . {~10 1;: 1 217 Figure 41. Cellulose Acetate Electrophoresis of Ceramide Trihexosidase, Form, A-l after Neuraminidase Treatment Electrophoresis was performed at room temperature and the strips were stained for activity. The strips contain the following: (1) ceramide trihexosidase, Form A-l obtained from affinity chromatography; (2) ceramide trihexosidases after a 1 hr incubation of Form A-l with neuraminidase; (3) ceramide trihexosidases after 4 hr incubation with neuraminidase; (4) ceramide trihexosidase, Form A-l, incubated 4 hr without neuraminidase; and (5) ceramide trihexosidase, Form B-V, obtained from iso- electric focusing. The strips were redrawn for purposes of photography. 218 219 .0000000000 0000000 000 00 00>0000 0300003 000>0000 000000000 000 0000000 0003 000 0.00 000000000 000 .00 00 00 me 000 00000000 0000030 0>00000030 0 00 0003000 003 .0-< 0000 .0000000000000 00000000 000000000 0000000000300 00 00 N 0000< 000000000 0000000000302 00000 .0-< 0000 .0000000000000 00000000 00 00003000 00000000000 .Nq 003000 220 (—).oz 0 Hd 3 .‘s 1 Fraction Number all. A ‘\ J! 1 40 41 7f l L J l O 8 8 9.2 ... u/pezKprKH 2—19 salowouoN (4422 Hd (+517st 221 is a sialoglycoprotein which can be converted to the asialo- glyc0protein, ceramide trihexosidase, Form B-V, by the action of neuraminidase. Preliminary Studies on Incorporation of Sialic Acid into Ceramide Trihexosidase, Form B-V A preliminary experiment was performed to determine whether there was any evidence for a sialyltransferase in porcine tissues which could convert human ceramide trihexo- sidase, Form B-V to Form A. Fresh porcine liver and kidney were homogenized in 0.25 M sucrose and incubated with the enzymes contained in whole plasma. The results of this experiment, indicated that both porcine liver and kidney homogenates could convert human plasma ceramide trihexo- sidase, Form B to Form A, although the kidney homogenate interconverted the proteins more rapidly. Incorporation of 114ClSialic Acid and [14C]N-Acetyl- glucosamine into Ceramide Trihexosidase, Form B Ceramide trihexosidase, Form B-V, obtained by isoelec- tric focusing, was incubated with [14C]CMP-sia1ic acid, using porcine kidney as a source of sialyltransferase. Three controls were run: one in which the basic protein was eliminated from the incubation to test for non-specific sialylation of other glyc0proteins in the incubation mixture; one in which the [14C]CMP-sialic acid was eliminated to test for spontaneous interconversion of the ceramide 222 trihexosidases; and one in which the [14C]CMP-sialic acid and the enzyme were incubated together in the absence of kidney homogenate to test for possible adhesion of radio- activity to the protein. Following 4 hours incubation with [14C]CMP-sialic acid fourteen proteins were formed, as shown in Figure 43. One of these proteins had the same electrophoretic mobility as ceramide trihexosidase, Form B-V and one of them had the same electrophoretic mobility as ceramide trihexosidase, Form A. In the control, containing no [14C]CMP-sialic acid, most of the protein was unchanged ceramide trihexosidase, Form B-V, although there were 4 other proteins of higher electrophoretic mobility. As shown in Figure 44 sialic acid was incorporated into all of the proteins except the most basic ceramide trihexosidase, Form B-V. Assuming an average molecular weight of 95,000 for all of the proteins, it was calculated that the protein of lowest electrophoretic mobility, into which sialic acid was incorporated, contained 3 moles of sialic acid per mole of protein. Thereafter there appeared to be a consecutive addition of 1 mole of sialic acid per mole of protein until the protein having the fastest electrophoretic mobility contained 15 sialic acid residues. These results should be interpreted with care, however, since the lower limits of accuracy for quantitating protein were used in some cases. In addition, the individual 223 Figure 43. Cellulose Acetate Electrophoresis of Ceramide Trihexosidase, Form B-V, following Sialyltransferase Treatment Electrophoresis was carried out at room tempera- ture and the strips were stained for activity. The strips contain the following: (1) ceramide trihexo- sidase, Form B-V obtained by isoelectric focusing; (2) ceramide trihexosidase, Form A-l, obtained by affinity chromatography; (3) ceramide trihexosidase after 4 hr incubation with [14C]CMP-sialic acid and kidney homogen- ate; and (4) ceramide trihexosidase, Form B-V, incubated 4 hr in the absence of [14C1CMP-sialic acid. The strips were redrawn for purposes of photography. 224 .11 - -‘aao. ‘ cum...“ M'".‘~ ' .0 ' ”—0-... -. m ~ 225 .00000000 000 0000 00000000 0000 000 00003 000000000 0003 00300> 00< .0000002 000 000000002 00 000000000 00 0000000000 0003 0000000 0: 000 000 000 >-m 0000 .0000000000000 00000000 0000 000< 00000m00 H 00 0000000000000 .00 003000 00 226 (C1) ugagmd b’rf SI § 9.. no no <1- (\I I T l I l r r | + - I INIIIIIIIIH <3 é 8 q (-) pewndmu! moo 3!|D!5-dWO[O,,] wdo 1 O O N 227 proteins would not have identical molecular weights. How- ever, it is safe to assume that the most acidic enzyme contains 15 i 4 residues of sialic acid. Since the [14C]CMP‘sialic acid was known to be unstable below pH 9.0, this experiment was repeated using [14C]UDP- N-acetylglucosamine, on the assumption that sialic aCid incorporation into the basis protein would follow the path- fl? way delineated for glycoprotein biosynthesis in liver (186). The pattern of radioactivity incorporated, shown in Figure 45, was basically the same as for incorporation of [14C]CMP- sialic acid although the quantity of the more acidic proteins was greater. The possibility that N-acetyl- glucosamine was incorporated as such into the proteins cannot be ruled out. This is not likely, however, since the electrophoretic mobilities of the proteins formed by incorporation of [14C]UDP-N-acetylglucosamine were identical with those of the proteins formed from [14C]CMP-sialic acid. In both experiments the total amount of radioactivity incorporated in the controls accounted for less than 5% of the total radioactivity incorporated into the proteins. 228 .00000000 000 0000 00000000 0000 000 M0000 000000000 0003 00000> 00< .0000002 000 00000000: 00 000000000 00 0000000000 0003 0000000 m: 000 000 000 >-m 0000 .0000000000000 00000000 0000 0000000000M00000<-20000H 00 0000000000000 .00 000000 229 (1:1) ugenmd brf 9or) no 0 N 11171 WWWV_ — — _ — — — — - — — — F — F - I J 5 O O l0 0 to N LO N (mnemmoow euwosoonlbweoo-Ndon m wdo DISCUSSION When ceramide trihexosidase activity was discovered in human plasma it was observed that there were two pH optima which suggested that two forms of ceramide trihexosidase f“" might be present in plasma. This possibility was confirmed i when it was found that the ceramide trihexosidase activity ' E at pH 5.4, called the A form, could be separated from the E activity at pH 7.2 (B form) by the low temperature ethanol fractionation procedure described by Cohn et al. (164). A combination of affinity chromatography and isoelectric focusing separated the A form of ceramide trihexosidase activity into two glyc0proteins which differed primarily in their kinetic properties, whereas the B form of the enzyme was separated into five proteins having distinct pI's. The occurrence of multiple molecular forms of plasma proteins is not uncommon. This phenomenon has been detected in many instances by electrophoretic and immunochemical studies, and the multiple forms of some of these proteins have already been isolated. For example, at least eight forms of plasminogen having different isoelectric points have been obtained by isoelectric focusing (187) and affinity chromatography (188), six forms of hypoxanthine-guanine 230 231 phOSphoribosyl transferase have been obtained by a combination of chromatography and isoelectric focusing (189), and a grOUp of seven liver 4-methylumbelliferyl-a- galactosidases related to each other by their sialic acid content have been reported (190). This multiplicity of proteins often arises from variations in the combination of several polypeptide chains, chemical modifications of #11 the completed protein, differences in the carbohydrate content of glyc0proteins, or genetic heterogeneity resulting from multiple loci or multiple alleles at the same locus ; 1 fg‘. coding structurally distinct polypeptide chains of the same protein (133). An understanding of the significance of the multiple forms of ceramide trihexosidase is particularly important since this enzyme might have therapeutic value in the treatment of Fabry's disease. This requires isolation of the individual molecular forms of the enzyme and characterization of their physical and biochemical properties. CHARACTERISTICS OF THE CERAMIDE TRIHEXOSIDASBS, FORM A Purity of the Enzymes The plasma ceramide trihexosidases obtained from Cohn fraction IV—l appeared to be homogeneous as determined by a constant specific activity following affinity chromatography and isoelectric focusing, by a single band on polyacrylamide gel electrophoresis, and by the presence of a single protein, 232 coincident with enzymatic activity, on isoelectric focusing and sucrose density gradient centrifugation. Although both proteins appeared to be homogeneous by all of the criteria employed, most glycoproteins show microheterogeneity. Alterations in the carbohydrate moiety which affect neither the enzymatic activity nor the charge on the protein probably would not have been detected by these techniques. More stringent criteria, employing larger quantities of protein, will have to be employed before the ceramide trihexosidases can be regarded as completely homogeneous proteins. At the present time these enzymes can be assumed to be 90-95% pure. Classification of the Ceramide Trihexosidases The results of neuraminidase treatment and sialic acid- incorporation studies strongly indicate that the A and B forms of ceramide trihexosidase are glycoproteins which differ in sialic acid content. Neuraminidase treatment of Form A-l ceramide trihexosidase converted it to a protein indistinguishable from B-V ceramide trihexosidase. [14C]Sialic acid and [14C]N-acetylglucosamine were incorp— orated into the B-V form of the enzyme forming a protein indistinguishable by electrophoretic mobility from the A forms of the enzyme. In addition all of the ceramide trihexosidases are stained by Schiff's-periodate on cellulose acetate strips. Direct proof of their classification can be obtained by sequencing the purified proteins. 233 If the completely sialylated form of ceramide trihexo— sidase contains 7-15 sialic acid residues attached to alternating carbohydrate moieties, it can be assumed that the ceramide trihexosidases are S-lO% carbohydrate. On the basis of the sialic acid incorporation studies it seems that the B-V form of ceramide trihexosidase is an asialoglycoprotein which can be converted into the A form of the enzyme by the action of a porcine kidney sialyl- transferase. Judging from the number of proteins which were produced by this method it is possible that plasma ceramide trihexosidase, form A, contains lO-lS sialic acid residues. This data supports the stepwise addition of sialyl residues to the B-V form of the enzyme. Studies on the properties of porcine serum and liver CMP-N-acetylneuraminic acid:g1ycoprotein sialyltransferases were reported by Hudgin and Schachter (191). The most effective acceptors for these enzymes were neuraminidase - treated al-glyc0proteins and sialic acid incorporation appeared to occur whenever a terminal galactose was linked (81+4) to a penultimate N- acetylglucosaminyl residue. These results should not be construed as an indication that the carbohydrate moiety of ceramide trihexosidase consists of repeating galactosyl- (Bl+4)N-acetylglucosaminyl residues, since most organ- specific sialyltransferases are a family of enzymes having different acceptor Specificities (192, 193). 234 The neuraminidase studies might also support the step- wise removal of sialyl residues resulting in 15 enzymes differing in their net negative charge. The number of intermediate forms of ceramide trihexosidase also can be explained on the basis of seven sialic acid residues by assuming that one of these residues determines the pH optimum at which the enzyme has catalytic activity. This speculation implies that the removal of a specific sialyl moiety causes the formation of a grOUp B protein which is consecutively desialylated to form B-V ceramide trihexosidase. Although there is no evidence in the literature which indicates that sialic acid plays a role in the catalytic site of an enzyme, it is feasible that the removal of a charged residue might cause a conformational change in the protein enabling it to be catalytically active at a different pH optimum. For instance, it is known that arylsulfatase A and B-N-acetylhexosaminidase A are converted to their respective B forms by neuraminidase treatment (95) and that a group of seven 4-methyl-umbelliferyl-a-galactosidases are converted to one electrophoretically slow component by neuraminidase (190). In addition, several biologically active hormones containing sialic acid are either partially or completely inactivated by neuraminidase treatment. These hormones include follicle-stimulating hormone, human chorionic gonadotropin and erythrOpoietin (194). On the other hand, the catalytic properties of several glyc0protein 235 enzymes are not effected by neuraminidase. These enzymes include serum cholinesterase, y-glutamyl transpeptidase, enterokinase and serum atropinesterase (194). General Pr0perties of the Ceramide Trihexosidases The two plasma ceramide trihexosidases are remarkably similar in their molecular weights, electrOphoretic characteristics, response to the lipid substrate under optimal conditions, and substrate specificity; they differ in their heat stability, solubility in butanol, and response to various inhibitors. The findings suggest that the two enzymes serve catalytic functions at different in vivo sites where their metabolic regulation may be partially controlled by the physiological environment. However it is difficult to attribute any physiological significance to the kinetic characteristics of these enzymes since little is known about either the substrate or protein concentration at a particular in viva site. Several sphingolipid hydrolases, including lactosyl- ceramidase (195), ceramidase (195) and the B-N-acetylhexos- aminidases (34, 144) are activated by detergents. This finding is usually explained as a requirement for an anionic detergent to form a molecular aggregate with a physical form suitable for hydrolysis of the lipophilic substrate (195). Likewise, the enhancement of enzymatic activity in the presence of specific salts (114) is normally attributed to an effect on the substrate environment. In the case of the 236 ceramide trihexosidases, these effects are more complex in the sense that detergents and salts may alter both the enzyme and the substrate. Effect of Sodium Taurocholate and Sodium Chloride on GL—3: The action of sodium taurocholate and sodium chloride on GL-3 is most easily explained in terms of their effect on the detergent micelles which solubilize this lipophilic substrate. In micelles composed of ionic monomers, the charged groups are arranged near the surface of the aggregate. This results in a high charge density and produces an electrical field which polarizes the surrounding solvent molecules (196). This type of molecular aggregate may have the ability to fix an enzyme on its surface. The addition of sodium chloride to this micellar system may effect the substrate in two ways. 1) It lowers the critical micellar concentration (CMC) of the bile salt (185). This enables the formation of additional micelles which increases the micellar surface area. 2) The addition of salts can alter the conformation of the Oligosaccharide which remains on the surface of the micelle (197). Both of these effects could make the substrate more accessible to the enzyme. Effect of Salt and Detergent on the Enzyme: As shown in Figure 26 A and B both forms of ceramide trihexosidase have sigmoid substrate saturation curves. Under the conditions of this experiment the substrate should exist in a mixed 237 micellar form with the bile acid at low substrate concentrations. Thus the sigmoid saturation would presumably not be due to formation of micelles but rather would result from an alteration of the mixed micelle or of the enzyme itself as the substrate concentration is increased. If the enzyme conformation is affected, it is not likely that the sigmoidal substrate saturation curve represents typical allosteric kinetics since the specific activity of the enzyme is unusually dependent on enzyme concentration (Figure 27). The following scheme is suggested as a working model to explain this dependency on enzyme concentration: Enzyme + micelle :: (enzyme—micelle complex) I product (Enzyme-micelle) + enzyme S (enzyme-micelle aggregate) complex This model assumes that the enzyme expressed its optimum activity when complexed with the mixed substrate-cholate micelle and that excess enzyme causes formation of an inactive enzyme-micelle aggregate. Since the formation of the postulated inactive enzyme aggregate does not appear to have precedence in the literature, some remarks regarding this process are useful. The active enzyme complex would presumably be dynamic in the sense that the enzyme, once complexed to a specific micelle, 238 either remains complexed to that micelle and interacts with several substrate molecules on its surface or is released free in solution after completion of one catalytic event prior to formation of the next enzyme substrate complex. The inactive enzyme aggregate could then reflect an interference with this dynamic process. Additional information regarding the formation of mixed micelles and enzyme-micelle complexes are required to describe this behavior in detail. Regardless of the lack of precedent for such a model, it is necessary to note that the description of these or similar micellar systems must include rigid control of enzyme concentration, ratio of enzyme to micelle concentration, and the type of salt employed. Analysis of this model reveals the following characteristics. 1) Any perturbation which affects the surface of the micelle or the number of micelles may alter the association of the enzyme with the micelle or the availability of substrate on the surface of the micelle, thereby affecting the specific activity of the enzyme. For example, the addition of potassium or ammonium salts which reduce the observed specific activity might be expected to alter the electrostatic surface potential of the micelle and interfere with association of the enzyme. Likewise, the addition of the neutral detergent Triton X-100 might be expected to reduce the surface potential and also affect the specific activity of the enzyme. 2) Addition of excess enzyme should lead to rapid inactivation or a sharp decrease 239 in specific activity as was observed (Figure 27). At low substrate concentrations the specific activity 7 M enzyme at which point remains constant up to 1.4 x 10' a rapid decrease in activity is observed. Assuming that a decrease in activity is due to the formation of an inactive enzyme aggregate, addition of sodium taurocholate and sodium chloride should increase the concentration of micelles and thus increase the range of protein concentration over which constant specific activity is observed. Addition of inhibitor at this substrate concentration also extends the range of constant specific activity either by increasing the number of micelles or by allowing formation of a higher concentration of enzyme in the complexed form by allowing more active enzyme molecules per micelle. At the intermediate concentration of substrate, the range of constant specific activity is greater than that observed at low substrate concentrations, but contrary to prediction the range of constant specific activity at this substrate concentration is not affected by sodium tauro- cholate or inhibitor. The significant increase in specific activity in the presence of inhibitor is not understood although this observation was made in one other experiment (Figure 28). At the high substrate concentration, addition of tauro- cholate extends the range of protein over which a constant specific activity is observed presumably by providing an .i‘ 240 increased concentration of micelles. The addition of inhibitor at this substrate concentration decreases the range of constant enzyme specific activity. Perhaps the combination of increased substrate and inhibitor concentration alters the cholate micelle surface allowing inactive enzyme aggregation at a lower concentration. “1 The range of constant specific activity with substrate ; alone at the high concentration is not increased over that observed at the intermediate concentration presumably because the substrate does not increase the number of micelles and the surface of the cholate micelle is already saturated. The addition of inhibitor at this substrate concentration now decreases the range of constant enzyme specific activity. Perhaps the inhibitor and increased substrate concentration alters the micelle surface allowing aggregation of enzyme at a lower concentration. The unusual effects of inhibition by digalactosyl- ceramide, that is, the stimulatory effects at low substrate concentrations and inhibitory effects at high substrate concentrations confirms the observation made by Ho with partially purified placenta ceramide trihexosidase (123). Contrary to her interpretation of the results in terms of an enzyme with an effector site, examination of Figure 27 shows that the sigmoidal substrate saturation curve should not be observed at low enzyme concentration. In other words, if the enzyme concentration is sufficiently low, the specific 241 activity in the absence of taurocholate is identical to that observed in the presence of taurocholate resulting in hyperbolic kinetics.' In view of the concentration dependency of the enzyme and the effect of various substances on this phenomenon, it should be pointed out that the inhibition studies conducted under a carefully standardized set of conditions may reflect alterations in the micellar system rather than classical inhibition kinetics. Classification by Gatt's Kinetic System: Since the hydrolysis of GL-3 is unusually dependent on the enzyme concentration it is difficult to attach significance to the "pseudo-micellar" system of Gatt. It is possible that the ceramide trihexosidases will hydrolyze some oligo- saccharides in the presence of lecithin. This suggests that the enzymes may be somewhat nonspecific exogalacto- sidases under the proper conditions. These experiments also indicate that the A-Z form of ceramide trihexosidase has a different mechanism for hydrolyzing oligosaccharides than the natural glycolipid substrate. According to Gatt's classifi- cation system (124) the A-Z form of the enzyme hydrolyzes trisaccharide equally well in the presence and absence of substrate micelles. However it should be noted that the concentration of lecithin is high enough to provide a mixed lipid-substrate micelle at all substrate concentrations. 242 Butanol Solubility The butanol method is normally applied with animal tissues to either solubilize the protein or to release the protein from the membrane. A list of several proteins obtained in solution or purified by butanol can be made. These proteins include actin, cholinesterase, lactic dehydrogenase and xanthine oxidase (165). Many of these .3 proteins are lipoprotein or glyc0protein in nature and sequencing of a few of them has shown that they contain a large number of basic amino acid residues. It appears that fits-u: ~. . butanol provides a hydrOphobic environment for the water- insoluble portions of these molecules. Most of these proteins are not completely butanol soluble and only xanthine oxidase was activated by butanol (198). However the CSS-iSOprenoid alcohol phosphokinase isolated from the bacterial membrane of Staphylococcus aureus is soluble and stable in water-saturated butanol (199, 200). This enzyme is a lipoprotein which has an absolute dependency for a phospholipid cofactor. However the phospholipid can be replaced by detergent in vitro and the enzyme is optimally active in the presence of 0.5 M sodium chloride. Unlike the ceramide trihexosidases, the reaction is linear with increasing concentrations of enzyme. 243 AFFINITY CHROMATOGRAPHY Protein purification by affinity chromatography utilizes the specific and reversible interaction of an enzyme with its substrate or a specific inhibitor. Purification is achieved by chromatographing a mixture of crude proteins on a column containing an enzyme—specific substrate or inhibitor attached by a covalent linkage to agarose beads. Proteins exhibiting affinity for the ligand will be retarded on the column to an extent related to their binding capacity for the ligand. The bound protein is eluted from the column by changing the elution medium so as to favor dissociation of the enzyme- 1igand complex. The general principles and specificity of this method were demonstrated by the use of specific adsorbents in the purification of staphylococcal nuclease directly from crude extracts, a-chymotrypsin, carboxypeptidase A, avidin, neuraminidase, and B-galactosidase (159, 162, 201). In most instances it is desirable to attach an enzyme- specific ligand to the matrix backbone to minimize the adsorption of non-specific proteins. This was the approach taken by Breslow and Sloan for purification of glucocere- brosidase, sphingomyelinase and arylsulfatase (94, 179). In the case of the ceramide trihexosidases, it was fortuitous that the specific ligand could not be attached to the matrix and that the available carbohydrate substrate had an (al+6) linkage. This probably aided in the adsorption of the .-_~_-i' 244 specific a-galactosidases since steric hinderance of the binding site was minimized. In all probability the ceramide trihexosidases would not have been separated by an affinity column adsorbent containing a trihexosylsphingosine ligand. There is no evidence to indicate that the ceramide trihexosidases have different affinities for the lipid substrate, whereas there are several indications that the enzymes have different affinities for the carbohydrate ligand. 1) Five of the seven ceramide trihexosidases bind to the affinity column with varying degrees of strength and are eluted in different fractions. 2) The A-l form of ceramide trihexosidase binds strongly to the column only in the presence of lecithin, but is only retarded in its absence. 3) Kinetic investigations employing the system proposed by Gatt (124) indicate that the two A forms of ceramide trihexosidase recognize different forms of carbohydrate substrates. a-Galactosidases of Whole Plasma Investigation of normal plasma revealed that the A forms of ceramide trihexosidase were present in nearly equal quantities. This indicates that some of the enzymatic activity was lost during the enzyme purification. It is likely that the ceramide trihexosidases are lost during Cohn fractionation since the specific activity of these fractions is 0.07 as compared to 0.2 in whole plasma. In addition, 245 100 ml of plasma contains as many pg of ceramide trihexo- sidase as are obtained from the amount of Cohn fraction representing 2-3 liters of plasma. Although there is no evidence, it is possible that most of the ceramide trihexo- sidase activity occurs as an inactive form in Cohn fraction V, since this is the fraction from which glycoproteins are classically isolated (183). The reason for their 1 inactivation is unknown but other experiments have indicated that an absence of enzymatic activity cannot be equated with an absence of the enzyme. ‘iw Digalactosylceramide:Galactosyl Hydrolase Normal plasma digalactosylceramide:galactosyl hydrolase activity is coincident with the activity of the non—specific a-galactosidase PN-A-Z. This enzyme is separated into two active components by isoelectric focusing and cellulose acetate electrophoresis. In Fabry plasma only the protein of slower electrophoretic mobility is detectable. Beutler and Kuhl recently reported that the normal plasma "ceramide trihexosidase" which hydrolyzes 4-methyl- umbelliferyl-a-galactoside consists of two isozymes distinguishable by electrophoretic mobility (121). They further reported that only the enzyme of slower electro- phoretic mobility was detectable in Fabry plasma and that it was indistinguishable from the normal plasma enzyme on electrophoresis. 246 The correlation between digalactosylceramide:galactosyl hydrolase and Beutler's 4-methylumbe11iferylgalactoside may be coincidental. However it is possible that other investigators are measuring GL-Zb hydrolase rather than ceramide trihexosidase. The studies of Crawhall and Banfalvi (122) correlate well with Beutler's studies, but the lack of detailed information in these reports forbids any definite conclusions. A deficiency of GL-Zb hydrolase explains the accumulation of this glycosphingolipid in Fabry kidney (49) and urinary sediment (182). It was previously assumed that ceramide trihexosidase would hydrolyze GL-Zb since the lipid has a terminal residue identical to that of GL—3. However, purified ceramide trihexosidase does not hydrolyze GL-Zb to an appreciable extent, although the lipid competitively inhibits both of the A forms of the enzyme. Non-Specific a-Galactosidases of Whole Plasma The question of why the artificial substrates can be used successfully to diagnose Fabry's disease when ceramide trihexosidase does not hydrolyze p-nitrophenyl-a-galactoside or 4—methylumbelliferyl-a-galactoside cannot be answered on the basis of present knowledge. However, it cannot be disputed that a depressed plasma a-galactosidase level for these artificial substrates is one of the biochemical characteristic of Fabry's disease. J’ 247 It is known that the artificial substrates are measuring the combined activities of a group of a—galacto- sidases whose specific functions are unknown. There are six detectable peaks of non-specific enzymatic activity obtained by affinity chromatography of whole plasma. The unexpected discrepancy of Fabry a-galactosidase activity before and after affinity chromatography is not easily explained. However it is known that enzymatic or electro- phoretic quantitation of total a-galactosidase levels often leads to erroneous assumptions concerning the level of a particular glyc0protein. For instance, the total glycoprotein level is depressed in renal diseases (202), cancer (203) and wounding (204), but the isolation and partial purification of specific proteins showed them to be 2- or S-fold elevated while other proteins in the preparation showed normal or slightly depressed activity (204). It might be postulated that an inhibitor of the non— Specific galactosidases accumulates in Fabry plasma and is removed by affinity chromatography. It is attractive to hypothesize that the accumulated proteins, presumably inactive forms of ceramide trihexosidase, are inhibitory to these galactosidases. However, the catalytically inactive proteins are not present in sufficient quantity to depress the total a-galactosidase activity. In addition, the Fabry heterozygote has nearly the same concentration of catalytically inactive proteins as the hemizygote but has a higher a-galactosidase activity. 248 A second alternative is that there is a depressed level of acidic a-galactosidases resulting from the renal complications of the disease. This is not a feasible suggestion because each Fabry differs in renal deficiency and the heterozygotes are more severely affected than the hemizygotes in some cases (205). The most practical suggestion is that an inhibitor of A unknown composition is present in Fabry plasma and causes partial inactivation of the non-specific a-galactosidases. Furthermore this inhibitor is removed by affinity chromatography, suggesting that it is not a-galactosidase in nature. This hypothesis derives some support from the fact that several ions are known to reversibly inactivate the ceramide trihexosidases. In addition, the plasma enzymes become rapidly inactive in whole plasma but the catalytic activity is regained by affinity chromatography. There is an unknown component eluted two to three fractions ahead of the first ceramide trihexosidase fraction in plasma containing no enzymatic activity. This component is yellow, non-protein in nature, and its color is destroyed by the addition of base. This may be only coincidental but these fractions are not obtained with fresh plasma which has catalytic activity at the time of chromatography. The addition of this component to active a-galactosidase fractions might determine whether it inhibits these enzymes. 249 GENETIC IMPLICATIONS OF MULTIPLE CERAMIDB TRIHEXOSIDASE DEFICIENCIES Fabry's disease has been shown to be an X-linked disorder by pedigree studies (59) and by the demonstration that clones of fibroblasts from heterozygotes display a bimodal population with respect to a-galactosidase activity (53). The nature of the enzyme deficiency could be explained on the basis of either an X-linked regulator gene or X- linkage of the structural locus for the enzyme. A structural gene mutation could be proved by demonstrating an abnormal residual a-galactosidase activity in the cells of patients with Fabry's disease. If no abnormal proteins were found, a regulatory mutation would be possible. The question of whether Fabry's disease involves a structural or a regulatory mutation has been considered in four recent papers (56, 190, 206-7). Beutler and Kuhl found that the 4-methylumbelliferyl-a- galactosidase activity of leukocytes and fibroblasts consisted of two a—galactosidases differing in electro- phoretic mobility (206). These proteins were designated as isozymes A and B, A being the more electrOphoretically rapid. The residual enzyme activity in leukocytes and fibroblasts of Fabry patients appeared to represent an increased quantity of the normal heat stable B form rather than an abnormal A form. Since these authors were unable to find an abnormal enzyme they entertained the possibility that the mutation was regulatory. 250 Nadler and Wood confirmed these findings but suggested that the enzymatic defect could be attributed to the absence of a specific sialyltransferase (56). This conclusion was made by analogy with Tay-Sach's disease and metachromatic leukodystrOpy in which the acidic isozyme is absent. This suggests that the basic form of the enzyme is the precursor of the acidic form to which it is converted by the addition of sialic acid residues. H0 et al. demonstrated similar findings in liver (190). However the liver a-galactosidases were separated into seven proteins by isoelectric focusing. On starch gel electrOphoresis three of these proteins migrated with the A isozyme, two with the B isozyme and two remained at the origin. Fabry liver contained only one B form of activity. Neuraminidase treatment of control liver supernates resulted in rapid conversion of the A forms to B forms of the a-galactosidase. Examination of the thermolability of the B enzyme following neuraminidase treatment revealed that control B activity was thermolabile while that from Fabry liver was thermostable. These authors suggested that Fabry's disease might be the result of a structural gene mutation or a specific sialyltransferase deficiency. Sutton and Omenn reviewed these papers and dismissed the possibility of a sialyltransferase deficiency on the basis that neuraminidase treatment of the a-galactosidases did not alter their catalytic properties (207). 251 In contrast to these findings it was found that the B forms of ceramide trihexosidase were absent in Fabry plasma while the A forms were present. In addition catalytically inactive proteins were found in hemizygous and heterozygous Fabry plasma. There is not enough information concerning the Fabry ceramide trihexosidases, human genetics, or the intermediary metabolism of glyc0proteins to enable a F33! J'L I reasonable hypothesis as to whether these enzyme alterations var ...-, are due to a regulatory or a structural mutation. However, the following suggestions are offered as an explanation for rm “0...; --. the ceramide trihexosidase pattern in Fabry plasma. It is possible that the A-l form of ceramide trihexo- sidase is the metabolically most significant form of the enzyme in viva and that the B forms are rapidly sialylated when synthesized in an attempt to regain a catalytically active form of this enzyme. It must be further assumed that interconversion of B to A in normal persons is controlled to maintain a dynamic equilibrium between the enzymes since normals have a constant ratio of A to B activity. Since the catalytically inactive proteins have a slightly greater binding capacity to the affinity column than the A-l form of ceramide trihexosidase, it is possible that they are partially sialylated B forms. However attempts to incorporate sialic acid into these proteins were unsuccessful. This suggests that these proteins are either fully sialylated or not recognized by the sialyltransferases. 252 If the second alternative is correct, there would be a defective glycosyltransferase resulting in the formation of a carbohydrate sequence not recognized by the available sialyltransferases. Alternatively, there could be an alteration at the catalytic site of the B form. In either case, an incomplete mutation must be assumed to explain the fact that the Fabry hemizygote has some protein which appears to be the normal A-l form in binding capacity to the affinity column and electrophoretic mobility. It is suggested that the A~2 form of ceramide trihexo- sidase arises from the A-l form. When the A-l form is desialylated, it is rapidly removed from the circulation, resialylated and sent back into the circulation as A-Z. This is suggested because the A-Z form of ceramide trihexo- sidase has the same electrophoretic properties as the A-l form, but is more susceptible to inhibition and may hydrolyze oligosaccharide substrates in a non-micellar system. These factors make it appear to be an altered form of a true glycosphingolipid hydrolase. In addition, whole kidney homogenate has A and B activity but the B activity rapidly disappears accompanied by an increase in A activity. Affinity chromatography showed this activity to be of the A-Z form. This suggests that there are two pools of B enzymes; one is newly synthesized and converted to the A-l form of the enzyme and the other is desialylated ceramide trihexosidase, Form A-l. 253 This might be disproved by studying the kinetics of the 4C]sia1ic acid into the enzyme formed by incorporation of [1 B form of the enzyme. Tissue culture studies might also be used to determine whether the B-V form of ceramide trihexo- sidase can be taken up by the organ, resialylated and sent back into the culture medium as a catalytically active protein having the kinetic properties of either of the A forms of ceramide trihexosidase. Although this scheme may be biochemically feasible it explains neither the genetics of the ceramide trihexosidases nor their relationship to the 4—methylumbelliferyl-a- galactosides. FEASIBILITY OF ENZYME REPLACEMENT THERAPY The results of plasma infusion experiments indicate that the ceramide trihexosidases have the capability of hydrolyzing GL-S in vivo and can be expected to decrease the level of accumulated plasma substrate. The enhancement of enzymatic activity in these experiments might have resulted from activation of the Fabry enzyme, but was more likely the result of several factors acting together to cause activation of the normal enzyme which was infused. When heparin is infused intravenously, there is a release of lipoprotein lipase from the vascular bed (208). This might result in the same type of activation observed after addition of butanol to crude enzyme preparations. Butanol causes a loo-150% activation in the activity of the ceramide trihexo- sidases. 254 The amount of enzyme infused in these pilot studies was not sufficient to indicate whether enzyme replacement would effect any clinical improvement in the disease. However, enzyme replacement by renal transplantation has indicated that the presence of catalytically active enzymes in Fabry patients will lead to the eventual control of the disease (209-10). The significance of the two primary forms of ceramide trihexosidase in plasma remains obscure. Attempts to determine the physiological role of these forms will be complicated since the B form is composed of several enzymatically active components. Certainly the question of their chemical and physiological relationship will have to be examined more critically when individual components are available in sufficient quantities for analyses of their carbohydrate composition and for studies of several kinetic parameters. The turnover rates in plasma of injected lysosmal hydrolases will be of considerable importance in the treat— ment of storage diseases by enzyme replacement therapy. It is not known whether there is a difference in the A and B forms of ceramide trihexosidase in this respect. However, recent information suggests that the presence of sialic acid residues on glyc0proteins prolongs their lifetime in circulation. It has been found that desialylated forms of some plasma glycoproteins can be removed from the circulation 255 by liver parenchymal cells (211). The binding of these proteins to the hepatic membrane was shown to involve the obligatory presence of sialic acid on what was presumed to be a glyoprotein acceptor site (212). In addition, it was suggested that this binding could be reversed by cyt0plasmic neuraminidase or changes in calcium ion concentration and pH (212). Perhaps the same mechanism will be involved in the removal of the B form of ceramide trihexosidase at a rapid rate if it is injected into patients or control subjects. It has not been determined whether binding of desialylated glycoproteins serves exclusively as a mechanism for cellular absorption and catabolism of circulating glyco- proteins, or whether these receptor sites might also be utilized for resialylation and subsequent release of reformed sialoglyc0protein into the circulation. Although studies on the turnover rates of the A and B forms of ceramide trihexosidase in plasma will be important in the choice of an apprOpriate form for therapeutic trials, another question is of equal importance to the ultimate therapeutic usefulness of lysosomal hydrolases in genetic storage diseases. Whether the catabolism of accumulated substances in abnormal cells can be achieved by injection of these enzymes depends on their accessibility to target organs. It is presumed that such cells will be able to form pinocytotic vesicles containing the hydrolase and that there will be transport in the cytOplasm to the secondary lysosomes 256 where stored material is located. It has been shown with cultured fibroblasts from Fabry patients that a plant a— galactosidase can decrease the amount of cellular ceramide trihexoside when added to the medium (213). It is difficult to predict which of the ceramide trihexosidases will be best able to penetrate abnormal cells in viva and which will have the longest intercellular lifetime. Another question to consider is whether the full compliment of a-galactosidases might have utility in the treatment of Fabry's disease. The Fabry patient has a depressed level of some non-specific a-galactosidases, GL-Zb hydrolase and the ceramide trihexosidases. The in vivo role of these individual enzymes has not been determined and it may be that some of the clinical symptoms of the disease arise from the absence of non-specific hydrolases. SUMMARY Plasma ceramide trihexosidase activity was separated into seven proteins. These glycoproteins consisted of two groups of enzymes related by their sialic acid content. The A form of ceramide trihexosidase was purified from Cohn fraction IV—l by a series of steps including ammonium sulfate precipitation, butanol treatment, acetone precipitation and affinity chromatography. The biochemical characteristics of the two A forms of ceramide trihexosidase were investigated. It was found that the two enzymes were remarkably similar in their molecular 257 weights, electrophoretic characteristics, response to the lipid substrate under optimal conditions, and substrate specificity; they differed in heat stability, solubility in butanol and response to various inhibitors. The ceramide trihexosidases were activated by sodium taurocholate and sodium chloride and their hydrolysis of GL-3 was non-linear with increasing enzyme concentration. An a-galactosidase affinity column adsorbent was prepared and used to study the enzymes in normal and Fabry plasma. It was found that normal plasma contained six non-specific a-galactosidases in addition to the ceramide trihexosidases. Affinity chromatography of Fabry plasma revealed that the A forms of ceramide trihexosidase were partially inactive, whereas the B forms were completely absent. There was also an accumulation of catalytically inactive proteins and an alteration of the specific activity of several of the non-specific a-galactosidases in Fabry plasma. Through the use of affinity chromatography a specific enzyme for the hydrolysis of GL-Zb was discovered in normal plasma. This enzyme was separated into two enzymatically active components by isoelectric focusing and cellulose acetate electrophoresis. Only the enzyme of slower electro- phoretic mobility was present in Fabry plasma. 10. 11. 12. 13. 14. 15. 16. BIBLIOGRAPHY Thudichum, J. L. W., Report Med. Off. Priv. Council, New Series, No. 3, Appendix 5, 113 (1874). Thudichum, J. L. 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Desnick, R. J., Simmons, R. L., Allen, K. Y., Woods, J. B., Anderson, C. F., Najarian, J. S., and Krivit, W., Surgery, 28) 203 (1972). Philippart, M., in Proceedings of the Symposium on Enzyme Replacement in Genetic Diseases, R. J. Desnick, W. Krivit, and R. Bernlohr, Eds., The National Foundation, New York, in press. Morell, A. G., Gregoriadis, G., Scheinberg, I. H., Hickman, J., and Ashwell, G., J. Biol. Chem., 246, 1461 (1971). Pricer, W. E., and Ashwell, G., J. Biol. Chem., 246, 4825 (1971). Dawson, G., Matalon, R., and Li, Y. T., in Proceedings of the Symposium on Enzyme Replacement in Genetic Diseases, R. J. Desnick, W. Krivit, and R. Bernlohr, Eds., The National Foundation, New York, in press. 1 1'1 APPENDIX 271 272 List of Publications Mapes, C.A., Anderson, R.L., and Sweeley, C.C. Trihexosyl- ceramide:Galactosy1 Hydrolase in Normal Human Serum and Plasma and its Absence in Patients with Fabry's Disease. Fed. Proc., 88, 409 (1970). Mapes, C.A., Anderson, R.L., Sweeley, C.C., Desnick, R.J., and Krivit, W.W. Enzyme Replacement as a Possible Therapy for Fabry's Disease, An Inborn Error of Metab- olism. Science, 169, 987 (1970). Mapes, C.A., Anderson, R.L., and Sweeley, C.C. Galactosyl- galactosylglucosylceramide:Galactosyl Hydrolase in Normal Human Plasma and its Absence in Patients with Fabry's Disease. FEBS Letters, 1, 180 (1970). Krivit, W., Desnick, R.J., Mapes, C., Anderson, R.L., and Sweeley, C. C. Recent Advances in Fabry' 5 Disease. Transactions of the Association of American Physicians, 83,121 (1970)—' Sweeley, C.C., Mapes, C.A., Anderson, R.L., Desnick, R.J., and Krivit, W.W. Fabry's Disease: Chemical and Enzymatic Abnormalities and a Pilot Study of Enzyme Replacement. In. Lipid Storag_ Disease- -Enzymatic Defects and C11nica1 Implications. J. Bernsohn and rossman (Editors), Academ1c Press, New York, 165 (1971). Sweeley, C.C., Mapes, C.A., Krivit, W., and Desnick, R.J. Chemistry and Metabolism of Glycosphingolipids in Fabry's Disease. In: S hin olipids, Sphingolipidoses and Allied DisordeFE. B. . 01k and S.MT Aronson (Editgrs), Plenum Publishing Corp., New York, 287 1972 . lg Press Mapes, C.A., and Sweeley, C.C. Properties of Ceramide Trihexosidase. In: Proceedin s 9: LEE 8 m osium on Enz e Replacement in Genet1c iseases. R.J. DesniEk, W. Erivit and R. Bernldhr (Editors) The National Foundation, New York. 273 Mapes, C.A., and Sweeley, C.C. Substrate Specificity of Ceramide Trihexosidase. FEBS Letters 8ggReview Mapes, C.A., and Sweeley, C.C. a-Galactosidases from Normal and Fabry Plasma. I. Preparation and Properties of an Affinity Column Adsorbent for Differentiation of Multiple Forms of a-Galactosidase Activity. 8. Biol. Chem. Mapes, C.A., Suelter, C.H., and Sweeley, C.C. a-Galactosi- dases from Normal and Fabry Plasma. II. Purification and Characteristics of Human Plasma Ceramide Trihexo- sidase (Form A). 8. Biol. Chem. Mapes, C.A., and Sweeley, C.C. a-Galactosidases from Normal and Fabry Plasma. III. Occurrence of Digalactosyl- Ceramide:Galactosy1 Hydrolase in Human Plasma. 8. Biol. Chem. Mapes, C.A., and Sweeley, C.C. a-Galactosidases from Normal and Fabry Plasma. IV. Interconversion of the Plasma Ceramide Trihexosidases. 8. Biol. Chem. "Tlfl’llflfiiuiflflfllflfljlllflifll'lflflfliflflflifllflfl