4.. .trrunu "4...: a ..ffimwfiggs‘ ... ..mwfg. 6 flaw} n a .Nngfiyanh . tinir. .5. i... m ..fi. .3% .r .1 -~.. GK: i N313 . i E NNLN L T ' I GANSTA c‘ ,- N." ‘WNLLAN 3H1 Lt’ .; I" .L. J . . ha... an; . L...‘.,§M....Lm #7. A) u .L . .- ... £25 Iii??? 3;? ,3... .2. -... Lax . .. w..i !.:...O.:,L§.rw H .3'.’ I This is to certify that the ‘ .. . ‘ thesis entitled STUDIES ON PHOSPHORYLATIVE PATHWAYS OF GALACTOSE METABOLISM IN RAT HEART AND BRA IN presented by WILLIAM DONALD LORNE MUS ICK has been accepted towards fulfillment of the requirements for Ph oDo _degree in BiOChemiStry Major professor . Date 4/5/77! ‘ " 0-7 639 ,77C) ABSTRACT I . C«\ STUDIES ON PHOSPHORLATIVE PATHWAYS ,fi\ OF GALACTOSE METABOLISM 1. IN RAT HEART AND BRAIN BY William Donald Lorne Musick The ability of adult rat heart to metabolize D-galac- tose via the sugar-nucleotide and reductive pathways was studied. Rat myocardial slices oxidized D-[1-1“C] galac- tose to 1“C02 at less than 5% the rate of D-[1-1“C] glu- cose oxidation. Furthermore, heart homogenates poorly con- verted D-[l-1“C] galactose into phosphorylated intermediates under conditions in which 25% of the labeled galactose was converted to metabolites by liver homoqenates. The Leloir pathway enzyme activities of rat heart were found to be present in amounts 8%, 38% and 23% that of rat liver for galactokinase, galactose l-phosphate uridyl trans- ferase and UDP-galactose 4'-epimerase, respectively. The corresponding values for the rat brain enzymes relative to liver were 27%, 11% and 120%, respectively. Michaelis con- stants for the three enzymes were comparable in heart, brain and liver. The reduction of galactose to galactitol in perfused heart preparations was dependent on the perfusate galactose William Donald Lorne Musick concentration and appeared to be substrate saturable with a "Km" of 30 mM for galactose. The rate of galactitol production in perfused rat hearts was several times greater than the rate of galactose oxidation to carbon dioxide by heart slices at equivalent perfusion and incubation media galactose concen- trations. Increasing the perfusate galactose concentration also had the effect of reducing myocardial ATP and creatine phosphate levels. Rat heart and brain, but not liver, crude enzyme prepa- rations produced what appeared to be galactose 6-phosphate in addition to galactose l-phosphate when incubated with high concentrations of D-galactose [30 mM]. Tissue hexo- kinase activities were measured as a possible explanation for galactose 6-phosphate synthesis. Galactose 6-phosphate was further identified in the brains of galactose-intoxicated chicks and rat hearts per- fused with galactose by spectrOphotometry and analysis by combined gas-liquid chromatography-mass spectrometry. Tis- sue concentrations of galactose 6-phosphate were compared to selected glycolytic and galactose metabolites. While galactose 6-phosphate levels were comparable to the levels of certain glycolytic metabolites, galactose 6-phosphate only comprised about 5% of the total galactose phosphate. The subcellular distribution of NADP+ and NAD+-dependent glucose 6-phosphate and galactose 6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose 6-phosphate William Donald Lorne Musick and galactose 6—phosphate with NADP+ or NAD+ as cofactor. While all of the tissues examined had NADP+-dependent glu- cose 6-phosphate dehydrogenase activity, only rat brain soluble fractions had NADP+-dependent galactose 6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose 6-phosphate dehydrogenase activities were kinetically different although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP+ or NAD+ cofactors but phosphatase activities hydrolyzing 6— phosphogalactonate, galactose 6-phosphate and galactose 1- phosphate were found in crude brain homogenates. The effects of galactose 6-phosphate and 6-phosphoga— lactonate on several glycolytic and hexose monophosphate shunt enzymes were investigated. 6-Phosphoga1actonate was found to be a competitive inhibitor of rat brain 6-ph03pho- gluconate dehydroqenase. STUDIES ON PHOSPHORYLATIVE PATHWAYS OF GALACTOSE METABOLISM IN RAT HEART AND BRAIN BY William Donald Lorne Musick A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1974 "Nature, my dear sir, is only a hypothesis." --Raoul Dufy ii ACKNOWLEDGMENTS The author wishes to thank Professor William W. Wells for his many helpful suggestions, thought-provoking discus- sions and unselfish financial support. Members of my guid- ance committee, Doctors John Wilson, Loren Bieber, James Schwinghamer and especially Steven Aust have my gratitude for their openness and encouragement. I would also like to gratefully acknowledge Doctor Joseph Prohaska whose thera- peutic Saturdays were largely responsible for preserving by sense of perspective. Finally, I wish to thank my wife Betty, whose love and support made these past years meaning- ful. iii TABLE OF CONTENTS Page List of Tables . . . . . . . . . . . . . . vii List of Figures . . . . . . . . . . . . . .viii List Of AbbreViations O O O O O O O O O I C C Xi INTRODUCTION Literature Survey . . . . . . . . . . l Mammalian Galactose Metabolism. . . . . 1 The Galactosemias . . . . . . . . . S Galactokinase Deficiency. . . . . . 6 Galactose l—Phosphate Uridyl Transferase Deficiency. . . . . . 7 UDP-Galactose 4'—Epimerase Deficiency . 11 Project Rationale and Objectives . . . . . 13 Organization. . . . . . . . . . . . 15 References . . . . . . . . . . . . 16 CHAPTER I. CHARACTERIZATION OF D-GALACTOSE METABOLISM IN RAT HEART . . . . . . . . . . . . 26 Abstract I O O O I O O O O O 0 O O 26 Introduction. . . . . . . . . . . . 27 Materials and Methods. . . . . . . . . 29 Animals and Materials. . . . . . . . 29 Tissue Preparation for Slice Studies. . . 3O Slice Incubation Procedure . . . . . . 30 Homogenate Preparation and Labeled Intermediate Studies . . . . . . . 31 Enzyme Preparations . . . . . . . . 33 Leloir Pathway Enzymes 33 Hexokinase . ._ . . . . . . . . 33 Enzymatic Assay Procedures . . . . . . 34 Galactokinase . . . . . . . . . 34 iv CHAPTER Page Galactose l—Phosphate Uridyl Transferase . . . . . . . . . 35 UDP—Galactose 4'-Epimerase . . . . . 36 Hexokinase . . . . . . . . . . 37 Determination of Acid Lability of Phosphate Esters. . . . . . . . 37 Heart Perfusions . . . . . . . . . 38 Metabolite Analysis . . . . . . . . 39 Resu1ts O O O O O O O O O O O O O 39 Oxidation of D-Galactose. . . .. . . . 39 Galactitol Production in Galactose- Perfused Rat Hearts. . . . . . . . 40 Production of Labeled Leloir Pathway Intermediates . . . . . . . 40 Enzymes of the Leloir Pathway . . . . . 41 Galactokinase . . . . . . . . 42 Galactose l- -Phosphate Uridyl Transferase . . . . . . . . . 42 UDP-Galactose 4'-Epimerase . . . . . 42 Evidence for Galactose 6-Phosphate in Heart and Brain Incubation . . . . 43 Hexokinase Activity of Rat Heart, Brain and Liver. . . . . . . 44 Effect of Galactose on Rat Heart Energy Reserves . . . . . . . . . 44 Discussion . . . . . . . . . . . . 44 References . . . . . . . . . . . . 50 II. IDENTIFICATION AND STUDIES ON THE METABOLISM OF GALACTOSE 6-PHOSPHATE IN HEART AND BRAIN O O O O O O O O O O O O O O 79 Abstract I O O O O O O O O O O O I 79 Introduction. . . . . . . . . . . 80 Materials and Methods. . . . . . . . . 82 Animals and Materials. . . . . . . . 82 Tissue Galactose 6-Phosphate . . . . . 83 Animal Treatment . . . . . . . . 83 Extraction of the Tissues . . . . . 84 Metabolite Determinations . . 86 Combined Gas-Liquid Chromatography- Mass Spectrometry . . . . . . . 87 CHAPTER SUMMARY Hexose 6-Phosphate Dehydrogenase Enzyme Preparations Assay Procedure. . In Vitro Synthesis of 6-Phospho-D- Galactonate . . . Synthesis of 6- -Phospho- D- Galactonic Acid . . Oxidation of 6- -Phospho-D- Galactonic Acid . . Galactose Phosphate Alkaline Phosphatase 6-Phospho-Galactonate Alkaline Phosphatase. Glycolytic and Hexose Monophosphate Shunt Enzyme Assays. Phosphoglucose Isomerase. Phosphoglucomutase. Glucose 6- -Phosphate Dehydrogenase 6-Phosphogluconate Dehydrogenase Soluble Hexokinase. Results . . . . . . Tissue Galactose 6-Phosphate Chick Brain Metabolites Galactose Perfused Rat Hearts Gas Chromatographic Analysis Mass Spectra. . . Hexose 6-Phosphate Dehydrogenase . In Vitro Synthesis of 6-Phosphoga1actonic Acid by Rat Liver Microsomal and Rat Brain Soluble Fractions Oxidation of 6-Phosphogalactonic Acid Galactose-Phosphate Alkaline Phosphatase 6-Phosphogalactonate Alkaline Phosphatase Effect of Galactose 6-Phosphate and 6- Phosphogalactonate on Glycolytic and Hexose Monophosphate Shunt Enzymes. Discussion . . . . . References . . . . . APPENDIX. vi Page 87 87 88 89 9O 91 92 93 93 93 94 94 95 95 96 96 96 96 97 97 97 99 100 100 100 101 101 105 133 138 LIST OF TABLES Table Page 1. Activities and Kinetic Parameters of Rat Heart, Liver and Brain Leloir Pathway Enzymes. . . . 53 2. Effect of Perfusate Galactose Concentration on Rat Heart Energy Reserves . . . . . . . . 54 3. Comparison of Selected Metabolites in the Brains of Chicks Fed Either a Control or Galactose- Containing Diet . . . . . . . . . . . 108 4. Comparison of Selected Metabolites in Rat Hearts Perfused with Media Containing Either Glucose or Galactose . . . . . . . . . . . . 109 5. Subcellular Distribution of NADP+ and NAD+-Depen- dent Glucose 6-Phosphate and Galactose 6-Phos- phate Dehydrogenase Activities in Rat Liver, Heart, Brain, and Chick Brain. . . . . . . 110 6. Hydrolysis of Tissue Galactose 6-Phsophate . . . 142 7. Recovery of [U-1“C] Glucose 6-Phosphate from TCA Extracted Tissue . . . . . . . . . . . 143 vii Figure 1. 2. 10. 11. 12. LIST OF FIGURES Metabolic Interconversions of D-Galactose . . Langendorf-type Rat Heart Perfusion Apparatus . Oxidation of D-[l-1“C] Glucose (A) and D-[1-1“C] Galactose to 1"C02 by Rat Heart Slices. . . The Effect of Perfusate Galactose Concentration on Perfused Rat Heart Galactitol Levels . . Production of Labeled Leloir Pathway Intermediates by Rat Liver and Heart Cell Free Homogenates. 'Reciprocal Plots of Galactokinase Activity in Rat Liver (A), Heart (B), and Brain (C) with Varying Galactose Concentrations . . . . . . . Reciprocal Plots for Rat Liver, Heart, and Brain Galactose l-Phosphate Uridyl Transferase Acti- vity with Galactose l-PhOSphate and UDP-Glu- cose as the Varied Substrates. . . . . . Reciprocal Plots for Rat Liver (A), Heart (B), and Brain (C) UDP-Galactose 4'-Epimerase Activity with Varying UDP-Galactose Concen- trations. . . . . . . . . . . . . The Effect of Elevated Galactose Concentrations on Rat Liver, Heart, and Brain Galactokinase ACtj-Vities C O O O I O O I O O O O Radiochromatography of the Products of Liver Ga- lactokinase Reactions Performed at 30 mM Galac- tose and the Effect of Alkaline Phosphatase and Mild Acid Hydrolysis on the Isolated Products Radiochromatography of the Products of Heart Ga- lactokinase Reactions Performed at 30 mM Galac- tose and the Effect of Alkaline Phosphatase and Mild Acid Hydrolysis on the Isolated Products Radiochromatography of the Products of Brain Ga- lactokinase Reactions Performed at 30 mM Galac- tose and the Effect of Alkaline Phosphatase and Mild Acid Hydrolysis on the Isolated Products viii Page 25 56 58 60 62 64 66 68 70 72 Figure ' Page 13. Hexokinase Activities and Distributions in Rat Brain, Heart and Liver. . . . . . . . . 78 14. Gas Chromatographic Separation of Galactose 6- Phosphate and Glucose 6-Phosphate Standards and Chick Brain Extracts . . . . . . . . 112 15. Comparative Mass Spectra of Chick Brain and Standard 8-D-Galactopyranosy1 6—Phosphate . . 114 16. A Kinetic Comparison of Rat Liver Microsomal and Rat Brain Soluble NADP +-Dependent Galactose 6-Phosphate Dehydrogenase Activities. . . . . . . . . .- . . . 116 17. Gas-Liquid Chromatographic Separation of the Trimethylsilylated Products of Rat Liver Microsomal and Rat Brain Soluble NADP+ - Dependent Galactose 6-Phosphate Dehydrogenase Activities. . . . . . . . . . . . . 118 18. Lineweaver-Burk plots of Rat Brain of Galactose 6-Phosphate,and Galactose l-Phosphate Phos- phatase Activities . . . . . . . . . . 120 19. Rat Brain 6-Phosphogalactonate Phosphatase Activity as a Function of 6-Phosphogalac- tonate Concentration . . . . . . . . . 122 20. Effect of Galactose 6-Phosphate and 6-Phospho- galactonate on Rat Brain Phospho-Glucose Isomerase . . . . . . . . . . . . . 124 21. Effect of Galactose 6-Phosphate and 6-Phospho- galactonate on Rat Brain Glucose 6-Phospha- tase Dehydrogenase . . . . . . . . . . 126 22. Effect of Galactose 6-Phosphate on Rat Brain Soluble Hexokinase . . . . . . . . . . 128 23. The Effect of 6-Phosphogalactonate on Rat Brain Phosphogluconate Dehydrogenase . . . . . . 130 24. Dixon Plot for the Ki Determination of 6-Phos- phogalactonate for Rat Brain 6-Phosphoglu- conate Dehydrogenase . . . . . . . . . 132 25. Hydrolysis of Galactose l-Phosphate and Galac- tose 6-Phosphate in 10% Trichloroacetic Acid . 145 ix Figure Page 26. Stoichiometry Between the Sum of Galactonate and Galactonolactone and Inorganic Phos— phate Released from Alkaline Phosphatase Treated 6-Phosphogalactonate . . . . . . . 147 27. Gas LiquidChromatography of 6-Phosphogalactonate and Alkaline Phosphatase Treated 6-Phospho- galactonate . . . . . . . . . . . . . 149 28. Gas Liquid Chromatography of 6-Phosphogluconate, 6-Phosphoga1actonate and Galactose 6-Phosphate . 151 29. Gas Liquid Chromatography of Galactonate, Galac- tonolactone and Galactose . . . . . . . . 153 ATP, ADP, AMP NAD+, NADH NADP+, NADPH UDP, UMP UDPG UDP-Gal Gal 1-P Gal 6-P Tris HEPES 6-P-Ga1a G6PD H6PD PPi TCA CPM ABBREVIATIONS Adenosine tri-, di-, or mono-phosphate oxidized and reduced nicotinamide adenine dinucleotide oxidized and reduced nicotinamide adenine dinucleotide phosphate uridine di- or mono-phosphate uridine diphosphate glucose uridine diphosphate galactose galactose l-phosphate galactose 6-phosphate tris (hydroxymethyl) aminomethane N-2-hydroxyethylpiperazine-N'~2- ethanesulfonic acid 6-phosphogalactonic acid glucose 6-phosphate dehydrogenase hexose 6-phosphate dehydrogenase inorganic pyrophosphate trichloroacetic acid counts per minute xi INTRODUCTION Literature Survey To supplement the discussion of the project's rationale and objectives, a background in mammalian galactose metabol- ism is presented in conjunction with a review of the human galactosemias. Mammalian Galactose Metabolism The sequence of metabolic steps by which D-galactose is utilized in mammals was largely elucidated by Leloir (1), Kalckar (2) and their co-workers. Initially, galactose is phosphorylated by a soluble galactokinase and ATP to form galactose 1-phosphate and ADP. Next, the uridyl portion of a uridine diphosphate glucose molecule is transferred to galactose l-phosphate yielding uridine diphosphate ga- lactose and glucose 1-phosphate. The enzyme catalyzing this reaction, galactose 1-phosphate uridyl transferase, is the second in the three enzyme system depicted in Figure 1. Following the coupling of galactose to a uridine nucleo- tide, uridine diphosphate glucose is regenerated from uridine diphosphate galactose by an epimerase-catalyzed inversion of the fourth carbon hydroxyl of the galactose moiety. The net result of the cycle is then the conversion of galactose to glucose 1-phosphate which can then be readily 1 2 assimilated through the glycolytic pathway. All of these enzymes have been described in a variety of mammalian tissues, such as liver (3-5), red cells (6-8), fibroblasts (9, 10), heart (11), brain and kidney (12-14). The impor- tance of the uridine nucleotide pathway in the metabolism of galactose can be appreciated by considering the heredi- tary deficiency of either galactokinase or galactose l-phos- phate uridyl transferase. Associated with both conditions is a reduction to about 20% of the normal capacity to oxi— dize galactose to carbon dioxide (15). Galactose 1-phosphate can also enter the sugar-nucleo- tide or Leloir pathway when activated with uridine triphos- phate by uridine diphosphate galactose pyrophosphorylase. This reaction, first described in liver by Isselbacher (16, 17) and since described in fibroblasts (18), involves the reversible pyrophoSphorolytic cleavage of UTP by galactose 1-ph03phate to form UDP-galactose and PPi (Reaction 4, Fig- ure 1). The activity of this enzyme relative to galactose 1-phosphate uridyl transferase varies considerably in dif— ferent tissues. In human fibroblasts the enzymes are of comparable activity (10, 18) while human liver transferase is about one hundred fold more active than the pyrophos- phorylase (19). Evidence that this enzyme is distinct from UDP-glucose pyrophosphorylase (Reaction 5, Figure l) is at best equivocal (18). Although the synthesis of UDP-glucose or UDP-galactose is reversible via the pyrophosphorylase enzymes, UDP-glucose and UDP-galactose can be hydrolytically 3 degraded to sugar monophosphates and UMP by a sugar nucleo- tide pyrophosphotase (Reactions 6, Figure 1). Uridine diphosphate glucose and UDP-galactose are not only inter- mediates of the Leloir pathway but are precursors to poly- saccharide, mucopolysaccharide, uronide conjugates and pro- tein- and lipid-based glycomacromolecules for example. While galactose is principally metabolized through the Leloir pathway, a number of auxiliary routes have been des- cribed. Galactitol, the reductive product of galactose, has been found in the tissues of patients with galactokinase and uridyl transferase deficiencies (20, 21). Rats or chicks fed large amounts of galactose also accumulate galactitol in their tissues (22, 23). The reduction of galactose to galactitol (Reaction 7, Figure 1) can be catalyzed by either aldose.reductase (24, 25) or L-hexonate dehydrogenase (26, 27) both of which are found in most mammalian tissues (28). Michaelis constants of 20 mM and 160 mM for aldose reduct- ase and hexonate dehydrogenase respectively reflect the low affinity these enzymes have for galactose (26). While ga- lactitol is not normally found in tissues, it does accumu- late when the intracellular galactose concentration approx- imates the Km of the reductive enzymes. Galactitol accumu- lation can however be prevented, if the tissues are first treated with tetramethylene glutaric acid, an inhibitor of aldose reductase (29). The subsequent metabolism of galactitol has not been extensively studied. Lens and liver polyol dehydrogenases, 4 which oxidize many polyols to keto sugars, do not utilize galactitol as a substrate (30). However, weinstein, §E_31, have reported an ATP, NAD+ and NADP+ dependent oxidation of labeled galactitol to 1"C02 by kidney and liver homo- genates (31). Since the rate of galactitol oxidation by the kidney was only about 3% the rate of galactose or sorbitol oxidation, oxidative metabolism of galactitol must be con- sidered insignificant. Indeed within twenty—four hours after the intravenous administration of radioactive galacti- tol to humans, as much as 99% is excreted unchanged in the urine with less than 0.5% of the label found in expired carbon dioxide (31). Cuatrecasas and Segal (32) have described an enzyme from rat liver which catalyzes the direct oxidation of ga- lactose to galactonic acid (Reaction 8, 9, Figure 1). Al- though the existence of this enzyme has been disputed (33, 34), the presence of galactonic acid in the urine of normal and galactosemic individuals (35) supports the presence of an in vivo oxidative pathway for galactose. The further metabolism of galactonic acid to D-xylulose through a B-keto galactonate intermediate has also been described (Reaction 10, 11, Figure l) but the evidence is equivocal (36). Xylulose could then be phosphorylated by liver xylulo- kinase (37) and subsequently metabolized through the hexose monOphosphate shunt (Reaction 12, Figure 1). In vivo studies with muscle phosphoglucomutase have demonstrated the conversion of galactose l-phosphate to 5 galactose 6-phosphate (38) (Reaction 13, Figure 1). Although the mechanism of this transformation is probably the same as for the glucose analogues (39), the reaction proceeds at less than 0.5% of the rate using glucose 1-phosphate as substrate. $013 and Crane (40) and others (41) have shown that brain hexokinase will phosphorylate galactose to ga- lactose 6-phosphate (Reaction 14, Figure 1) but at a much slower rate than glucose phosphorylation. Galactose 6- phosphate, once formed, could become substrate for a liver microsomal dehydrogenase to form 6-phosphogalactonic acid (Reaction 15, 16, Figure 1) (42). Subsequent oxidation of 6-phosphoga1actonate to pentose phosphates has not been demonstrated (Reaction 17, 18, Figure 1) (43). The isomerization of galactose 6-phosphate to tagatose 6-phosphate is known to occur in bacterial systems (Reaction 19, Figure l) (44). While this isomerization has not been demonstrated in higher organisms, beef brain preparations will rapidly convert tagatose 6-phosphate to triose phos- phates, presumably with tagatose 1, 6-diphosphate as the intermediate (Reaction 20, 21, Figure l) (45). The Galactosemias Each of the enzymes of the Leloir pathway are now known to have at least one associated genetic abnormality in man (46). Since the literature on the galactosemias is exten- sive and excellent reviews are available elsewhere (46, 47) only a brief summary of some clinical, genetic and biochemi- cal aspects of the diseases will be presented here. 6 Galactokinase Deficiency. The first recognized case of galactokinase deficiency was reported by Gitzelmann in 1965 (48). The only clinical manifestation of the disease is the appearance of cataracts within the first few months of life (49). While a few patients with galactokinase de— ficiency have been described as having neurological disorders (50, 51), it is not considered a characteristic of the dis- ease (50). The onset of lenticular opacities, which if un- checked can lead to blindness, is prevented or even reversed if galactose is omitted from the diet (52-56). Since the develOpment of cataracts is not apparent for several months, early detection of galactokinase deficiency is dependent on routine screening of blood and urine for galactose. Shortly after the ingestion of galactose as lactose, galac- tose is found in the urine and moderately high concentra- tions are found in the blood, about 5 mM (52, 56). Smaller quantities of galactitol (20, 51, 56) and galactonic acid (51) are also excreted in the urine following galactose ingestion. Galactokinase deficiency is inherited as an autosomal recessive trait (56, 57). Estimates of the occurence of the homozygous condition range from 1 in 46,000 to l in 84,000 depending on the population analyzed (57, 58). The hetero- zygote has about one-half normal levels of galactokinase, and except for the occasional appearance of cataracts are asymptomatic (54, 59, 60). The loss of galactokinase activity in the tissues of 7 the homozygote is believed to be complete (51), although uridyl transferase and epimerase activities are normal (20, 50, 52, 56). Despite the lack of this enzyme, galactose can still be slowly oxidized to carbon dioxide (51). Since these patients oxidize C-1 of galactose at a greater rate than C-2 and since galactonic acid is found in their urine, a direct oxidative pathway for galactose would seem to be functioning in vivo. Studies on the incidence of galacto- kinase deficiency in persons developing cataracts before the age of forty have revealed a rate of occurence of about 1 in 50 (61). Cataractogenesis in galactokinase deficiency is believed to be related to galactitol accumu- lation in the lens (47). In vitro studies have shown that galactitol accumulation and lenticular opacities can be prevented if the tissue is first treated with tetramethylene glutaric acid, an inhibitor of aldose reductase (29). Galactose l-Phosphate Uridyl Transferase Deficiency. This disorder, also known as classic galactosemia, is the most severe of the galactosemias. It is readily detected within the first few days of birth by poor appetite, weight loss, vomitting and diarrhea. Secondary liver involvement as evidenced by hepatomegaly or jaundice results if galac- tose is not removed from the diet (62). Cataracts have been reported shortly after birth (63) but generally develop after several months (62). Mental retardation of varying degrees (62, 64, 65) is a common feature of the disease if galactose ingestion is unrestricted. In the extreme, 8 continued galactose administration can lead to seizures and death. Other symptomology of classic galactosemia is reviewed elsewhere (47, 62). As in galactokinase deficiency, galactose, galactitol and galactonic acid are found in the urine following the consumption of galactose (35). However, in marked contrast to galactokinase deficiency, classic galactosemics are characteristically hypergalactosemic [blood galactose greater than 20 to 30 mM (66, 67)] and frequently hypoglycemic (62). The disruption in phosphate ester metabolism originally described by Schwarz (7), is believed to be a result of the accumulation of galactose l-phosphate in the tissues (7, 68, 69). Classic galactosemics given an oral galactose toler- ance test rapidly accumulate galactose l-phosphate in their erythrocytes (70). Galactose l-phosphate has also been found at autopsy in liver, kidney, brain, heart, tongue and adrenal gland (71). Another feature in common with galac- tokinase deficiency is high levels of tissue galactitol (21, 72, 73) which is also believed to be related to cataract formation. Classic galactosemia is also an autosomal recessively inherited disease (74-76). The most recent estimate of its frequency of occurence is l in 45,000 (77). The hetero- zygote has one-half normal levels of erythrocyte uridyl transferase and is clinically healthy except for occasional minor symptomology on heavy galactose loading (62). At this point it is useful to classify the variants of 9 classic galactosemia into two categories based on clinical symptomology. The first variant of classic galactosemia, the Duarte variant, was described by Beutler and co-workers in 1965 (78). The Duarte gene is allelic with the galac- tosemic gene and is also autosomal recessively transmitted (79-81). Erythrocyte uridyl transferase activity of the Duarte homozygote is about 50% of normal and about 75% for the heterozygote (78). Starch-gel electrophoresis of Duarte homozygote erythrocyte transferase reveals a three-banded pattern (82), whereas the normal gives a single band. The Duarte variant is not associated with any disease state or clinical problem. Another variant exhibiting a three- banded electrophoretic pattern [but with a different dis- tribution of activity amongst the bands] and no clinical symptomology has been recently described by Ng, §E_al. (83). The homozygote of this variant [The Los Angeles variant] has about 150% the normal activity of erythrocyte transferase and the heterozygote has about 110% of the normal. Mixed genotypes of Duarte, Los Angeles, galactosemic and normal individuals have also been described (83). Another slow-moving electrophoretic form of transferase, termed the Rennes variant, was reported by Schapira and Kap- lan.(84). This mutant is characterized by about 7% of normal erythrocyte transferase activity. An unstable transferase [Indiana variant] associated with an incomplete loss of ac- tivity has also recently been reported (85). Both the Rennes and Indiana variants show the clinical symptomology of 10 classic transferase deficiency. A third mutant, the Negro variant, has no erythrocyte transferase activity but does have about 10% of normal activ- ity in the viscera (86-88). Patients with this disorder are able to metabolize intravenously administered galactose to carbon dioxide as well as normal individuals (15). Inter- estingly, the Negro variant also suffers the same symptomol- ogy as the classic galactosemic (90). Despite normal levels of galactokinase and UDP-galac- tose epimerase in the classic galactosemic (9), the loss of transferase activity is complete (91). Immunologic studies have shown that the uridyl transferase gene product is synthesized but it is not enzymatically active (92). As in galactokinase deficiency, classic galactosemics are able to slowly oxidize galactose to carbon dioxide (15). Again, C-1 of galactose is oxidized faster than C-2 (89) and since galactonic acid is present in the urine, a direct oxidative pathway for galactose seems to be operating. Other mechanisms have been proposed to account for the residual galactose metabolism, notably the synthesis of UDP- galactose from UTP and galactose 1-phosphate via UDP-galac- tose pyrophosphorylase (16), thus bypassing the uridyl trans- ferase reaction. Since this bypass cannot occur in the ga- lactokinase deficient individual, and since the extent of galactose oxidation to carbon dioxide is similar in both diseases (51), the direct oxidation of galactose is a more likely alternative pathway in classic galactosemia. 11 The molecular events underlying the complex symptomol- ogy of uridyl transferase deficiency are not completely understood. While it seems certain that the accumulation of galactose l-phosphate is involved, the exact mechanisms are unknown. Studies by Pennington and Prankerd (66), Mayes and Miller (93), Ng (94), Donnell et_al. (95) and Wells (96) suggest that galactose l—phosphate may be involved in a futile cycle of ATP hydrolysis leading to a reduction in cellular energy reserves. Also, since the blood galactose levels encountered in uridyl transferase deficiency are many times higher than in galactokinase deficiency, galac- tose competition for glucose entry into the cell has been proposed as a factor contributing to the etiology of clas- sic galactosemia (97, 98). Other mechanisms, such as the inhibition of normal enzyme systems by galactose l-phos- phate (99-101), hyperosmolar dehydration of nervous tissue (102), increased fragility of neural lysosomes requisite to the release of acid hydrolases (103) and disruption of phospholipid metabolism (104) have also been suggested. UDP-Galactose 4'-Epimerase Deficiency. This abnormal- ity, only recently discovered by Gitzelmann (65), has only one known homozygote. The condition was accidentally dis- covered during routine blood galactose screening. What initially seemed to be elevated blood galactose was later determined to be galactose l-phosphate. Subsequent analysis of erythrocyte galactokinase and uridyl transferase revealed normal levels of these enzymes. In vitro studies with the 12 patients erythrocytes showed only insignificant metabolism of 1"C-labeled galactose or UDP-galactose to 1|’COz but ready conversion of 1"C--1abeled UDP-glucose to 1"C02. Epi- merase deficiency was then proved by demonstrating the in- ability of erythrocytes to convert l"C-labeled UDP-galac- tose to ll'C--labeled UDP-glucose. Analysis of the red and white blood cells showed no epimerase activity although cultured liver and fibroblast biopsy specimens show normal epimerase levels (106). When galactose tolerance tests were administered, blood galac- tose was only mildly elevated [approximately 1 mM] whereas erythrocyte galactose l-phosphate was markedly increased to levels similar to those seen in classic galactosemia. Urinary excretion of galactose and galactitol following milk ingestion was insignigicant although a mild galacto- semia was observed following tolerance tests. A study of the family of the patient revealed indi- viduals with only half normal erythrocyte epimerase levels. When these persons were considered heterozygous for epimer- ase deficiency, a clear recessive inheritance pattern was seen. To date [the child is now two years old], no clinical or biochemical pathology has been associated with the defi- ciency. Since the loss of epimerase activity seems to be restricted to the blood tissues, multiple forms of epimer- ase coded by more than one gene locus would seem to be in- dicated. Indeed, multiple forms of mammalian liver and erythrocyte epimerase have been reported (107, 108). 13 Project Rationale and Objectives Biochemical studies on the pathogenic mechanisms in- volved in classic galactosemia have relied on the use of model systems. The newborn chick has been useful in this regard since a debilitating neurologic syndrome, believed similar in nature to the human disease, can be produced by the dietary administration of large amounts of galactose (109). Under these conditions, the rate of galactose influx exceeds the metabolic capacity of the tissues. The result is an accumulation of sugar nucleotide and reductive path- way intermediates, much the same as in uridyl transferase deficiency (23, 96, 104, 110). Despite the success of the chick as a model, a sys- tem more closely allied to man would be desirable. The only mammalian system studied to date has been the rat (111- 115). Unfortunately, the rat is relatively resistant to the toxic effects of galactose loading (116) presumably due to an active liver galactose metabolism (117, 118). However, studies by Tygstrup and Keiding (119, 120) have shown that ethanol will greatly increase the susceptibility of rats to galactose toxicity. Ethanol administration increases the hepatic NADH/NAD+ ratio which is inhibitory to UDP-galactose epimerase (121, 122). Again, an accumulation of the Leloir pathway intermediates results (123). Since the success of this model depends on altering the intracellular redox state and has a secondary effect of trapping uridine phosphates (123) [effects which galactose alone does not produce], this 14 approximation of the galactosemic state must be regarded with skepticism. Isolated mammalian organ systems have not been used for the study of galactose toxicity although galactose metabol- ism in various rat tissues has been investigated. Since the ability to metabolize galactose varies considerably in rat tissues (118, 124), the possibility of using a single organ to mimic the galactosemic state was investigated. Previous work by Fisher and Lindsay with perfused rat hearts estimated that galactose utilization [determined as non- glucose reducing substance] was no more than 5% of glucose utilization (125). Furthermore, Quan-Ma and Wells had shown that rats fed galactose-enriched diets accumulate large amounts of galactitol in cardiac tissue (22). These observations, along with the fact that heart preferentially utilizes ketone bodies and fatty acids to carbohydrate fuels (126-129), suggested that an active galactose metabolism may not occur in the rat myocardium. Since isolated meta- bolic studies could readily be performed with perfusion systems, a rat heart model for studying the toxic effects of galactose was particularly attractive. Accordingly, studies were undertaken to investigate the mechanisms of galactose metabolism in rat heart. Initial experiments were performed to determine the overall ability of cardiac tissue to dispose of galactose via oxidative or reductive pathways. Subsequent experiments were designed to corre- late the kinetic prOperties of the Leloir sugar nucleotide 15 enzymes with the observed patterns of galactose metabolism. Throughout these experiments, selected comparisons were made with rat liver and brain. During the course of these investigations, evidence for an alternate 6-phosphorylative pathway of galactose metabolism in rat heart and brain was obtained. Since such a pathway might further the understanding of the residual galactose oxidation observed in galactosemics, the last portion of this work is concerned with the study of this pathway in heart and brain. Organization The body of the thesis has been divided into two sec- tions, each following the format used in most biochemical journals. Portions of both sections have been accepted for publication in Archives of Biochemistry and Biophysics and are currently in press under the title "Studies on Galactose Metabolism in Heart and Brain: The Identification of D-Galac- tose 6-Phosphate in Brains of Galactose-Intoxicated Chicks and Rat Hearts Perfused with Galactose" by W. Donald L. Musick and William W. wells. A second manuscript dealing with material from the second section titled "Studies on Galactose 6-Phosphate Metabolism in Rat Heart and Brain" has also been submitted, under the same authorship, to Archives of Biochemistry and Biophysics. An appendix has been in- cluded to detail methods and results not adequately described in the text. lo. 11. 12. 13. 14. 15. 16. REFERENCES Leloir, L. F., (1951) Arch. Biochem. Biophys., 33, 186. Kalckar, H. M., Braganca, B. and Munch-Peterson, A. 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(1972) Cardiology, 31, 51-54. ll [Ill-Ill 'l‘l [I'll-I‘l- lI'l 1' ' t. .Ama.~.H.NI.U.mv mmmaooam .HN “AHH.H.S.~ .U.mv ommswx oumnmmonmlm mnouosum .om “A .H.m.m .U.mv ommnme Iowa muwcmmonouw mmouomHmm .mH “onwahxonumomc owns uwcouomammosmmonmlm loamxlm .mH “A .H.H.H .U.mv mmmcmmoucanmp owns owsouomamm03mmonmlw .hH “A .H.H.m .U.mv mmmcouoma maouomalw msouomammosmnonmlm .mH «A H.H.H .U.mv mmmcmmoucmnoo mumnmmonmlm mmoxm: .mH «AH.H.S.~ .U.mv omwswxoxmn .qH “Aa.m.e.m .o.mc ammussoosamoammonm .mH «AFH.H.F.~ .onmc mmmcaxoflaasx .NH mommamxooumomp cfiom oasouONHmm oumxlm .HH «A .H.H.H .O.mv mmmcmm Iouomnop meow oesouomamm .OH “A .H.H.m .U.mv mmmsouoma osouomalm ocou nomamm .m “Ame.a.a.a .o.mc mmmammounmamc mmouomamm .m limH.H.H.H .o.mc mmmsmmouomnmo mumcoxmnlq no AHN.H.H.H .U.mv mmmuoscmu omOpHm .5 “AI.H.m.m .O.mv ommuonmmonmoumm mmouomammlmos no omoosamlmoa .w «Am.h.h.m .U.mv mmmamuonmmonmouam mmoosamlmob .m “Aoa.h.h.m .U.mv mmmamu Ionmmocmouhm wmouomammlmoo .¢ «A~.m.H.m .U.mv mmmuwfiflmml.v mmouomamm umoo .m “ANH.>.>.N .O.mv mmmummmsmuu Hmcflus mumnmmonmla mmouomHmm .N “Am.a.h.m .O.mv mmmsflxouomamm .H "mum msowuommu oouonadz .mmafimm Iouomamm on» pg mumasasoom ou c3osx omonu mum mmuwaoomuos conflaumpco 24 .mmOHUMHMUIQ HO mGOflmeerOUHmUGH OHHOQMfiwz .H musmflm 25 macaque mo onouosum mat AB< mum 30153? 2 04.2 OdGOOQHU nab ouoxuu unfit! +mn¢2 32 odaooadu-mm .1 osououale 9338.3 Email +mnaz «no mmouosuh Into onHoosHU oao< oucousonHUImoD mud uuoosao/ onoonaonmoa 17!, and H mum god—Aux jl ocean—Aux «00 gas mat «00 And. .Ha. cans egos cacouooanu cacouunauu concuua umu ouoxua ouoxua manta mac: .Hac .sa. .oa. +gn coumxomum .2 .m .m samuoum mE\:wE\UoBHOM poacoum noaoss no commoumxm ma Muw>wuoda Awe emo.o“v~.a Ace mmo.o«am.a .sufl>auo« Ave moa.ouem.m ma.o «H.c mo.o ha.o as .ss chasm assumes saunas: and awe omouomamu mumuumnsm Ame mmo.oflmm.c Lac mvm.ofima.v .mc mmo.oamvo.o «sua>auo< «H.o m~.o 5H.o sa.o as .sx upmmm Hmoamao sausage mud Haw . omouomdmo mumuumnsm Ame mea.ouwm.~ am. muo.oflom.oa Ame hma.o“om.m «suw>auo« o~.o mH.o ma.o ea.o as .ss po>aq Hmoumos saunas: mua Hmo mmouomamo mpauumnsm mmmuoaflmm|.¢ mmmuommsmua Havana mumswxouomamo commas mmouomamoamas m|H_wmouomam0 .mmEhucm hmznpmm Hwoamq samum one Hm>aq .uumom pom mo muouufimumm oaumcwx can mmfiuw>wuo¢ .H manna 54 Table 2. Effect of Perfusate Galactose Concentration on Rat Heart Energy Reserves. Perfusate Galactose Concentration (mM) ATP Creatine Phosphate pmoles/gm tissue 2 2.72:0.43 2.92:0.68 5 2.0510.36 2.30:0.62 10 2.16:0.68 1.87:0.80 15 1.9510.52 2.0610.02 Values represent duplicate determinations on 4 hearts 1 S. D. Figure 2. 55 Langendorf-type rat heart perfusion apparatus. Components are as labeled: (A) constant pressure perfusion pump; (B) perfusate reservoir and oxy- genation chamber; (C) gas hydration chamber; (D) aortic bubble trap with attached mercury mano- meter; (E) aortic cannula and mount; (F) gravity- fed washout reservoir; (G) constant temperature circulating water bath. .-_- ..‘nh —_.F. — _.-w —._—— — “—I‘ —.-. 56 57 .Amv mucmfiflpmmxm o no Adv mucmfiaummxm v mo coau8fl>m© oumccmum H some on» musmmmummu ucfiom comm .cofluoom mpozumz can mamwumumz mnu CH confluomwp mm cmmmmuu mm3 Ummmwamu ~00 .Uohm um "HO: m.oa mmouoma -mo _o.~uaauo 2: mm no ”so: m.ol mmoosfim _o.snaauo zs m manuam can .v.s mm .ummmso mumnmmonm Hmmcflmlmnmnm HE o.m msflswmucoo mxmmHm Headacmaum HE om ca coumnsocw mumz ammo: you mo Hunmflo3 who .mE om waoumEonummmH mmowam mouse .mmoHHm ammo: new we Noo.s on Ame mmopomflma Ho._-HL-o cam lac mmoosam _u.~ua_na mo coaumeflxo .m musmflm 58 l J noxoaonooaa '00,, snow L O A usfaaonooua'h a” sa'loufl' O 90 |20 [50 60 I5 30 60 90 IZO ISO 30 TIME (minutes) TIME (minutes) 59 Jul. lull" I'll 1| iii .muuwms v MOM .o .m H some may usmmmumou mmsHm> .Hmconumz can mamaumumz coma mamaamcm muaaoomuoe How mmcou ooaooo cmmouuwc ofiswwa nufl3 pomEmHo oumoum wum3 hummus .ooflumm coflmsmuom may mow3odaom .mmouomHmm :5 ma no OH .m .N Hmcuflm can omoosam SE N msflsflmusoo finance soflmdwnmm m nuw3 msumummmm sofimsmuom mmwuumpocsmmcmq m ca mouscwfi on you ommsmumm mum3 mpumoz pom .mam>ma Houfiuomamm ammo: umu consumed so coflumuusmocoo omouomamm mummsmumm mo uommmm one I! {l lcl‘ll 1' II I ll.‘ [I'll ‘ .v mufimwm 60 $2.... mmoeofifia animal—mm.— O— n d I I I I I I I I I I 4 q 0 ll " — 0' O EWHD’IOIIIOV'IVD] 5108811 I: ’2 O ( III '| I l 11 l ! l‘l All. .‘I l I. 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L 1 O.~ 10.0 .. of l o .n > > > n... 1.! .... . od 1 o s U m 4 67 .mwmxamse Housmfioo we cwcflmuno mum3 mmcwa unmwmuum yam umom .cwmuoum mE\cwE\uoocoum mmaoes mm ommmmHQXm mum mwfluwoon> .HE ~.o mo oEdHo> Hmcfim m on nouns can .GOAPmHmmmum mfihucm mo H: om msowumnuswocoo mcflmum> um mmouomHmm nu HIDE nan: .st some o.m mm .mumcaosHm eaaeom ..zs m.~a +n nuw3 Muw>wuom mmmnwEHmml.v omouomammnmob AOL nacho new .Amv ammo: .Amv Hm>wa you Mom muon Accommwomm .m musmflm 68 a: 333.33 Iluflsll n 4 1 4 ‘ 69 .cowuoom moonumz one nawfipoumz on» Ca confluommc mm omfiuomuwm oum3 magnum one .HE H.o mo mesao> Assam m nuw3 m musmwm mo omonu mum3 mCOAUHocoo momma .meaumuucmosoo omouomamm mswaum> auw3 mmfluw> Iauom omwsfixouomamm camps new .uummn .Ho>fla Mom uoam avaooam>noumuunnsm d .mowuw>wuom mmmcwxouomamm camps can .uummn .Hm>wa you so mcowumnucmocoo owouomamm poum>oam mo uommum one .m musmflm 70 J1 If r I Z " :- é a: m < LIJ I K m 2 ...I 1 J/ 1 If 0 o '0 (nymmd Ew/ugm/sonpom sqowu) AllAllOV BSVNIXOIOV'WO IS 20 25 30 [GALACTOSE] (m M) IO 71 .mwumnmmonmocoa mmoan one mmoxw: on mcflccommmuuoo EmHmOPMEoHno may no msoflmmu on» muonop msouum HMOflun> one .sowuowm moonumz can mamanmumz on» CA pwnwuomoo mum mmmhaouohn been one .owumESNcm .asmmumoumfiouco mo mHHmumo .pmnmmumoumeownomu can gunman man so saonm mm couponu .mnmmumoumsouno women an pwumaonw oum3 mucopoum m>Huomowcmu one .HE H.o mo mesao> Assam m :uw3 .w musmflm aw cmcwauso no _HUn NH omouomHmmno SE on no omeuounmm mums Hm>wa How mammmm mmmswxouomamo .muosooum omumaomw on» so mammaouoan neon page one mmmumnmmosm mcflamxam mo uomumm may one mmouomflmm :8 on no UQEMOMHmm mcoauommn mmmcflxouomamm um>HH mo muosconm on» no hammumoumeonnoowpmm .oa whamwm 72 2.02.0 10¢». 20 0. o 0 1 1 fi 1 1 J 1 q d 1 4 d *m moxm...‘ Idfiafiuqdfiq 2.282: 33 2.2+ 32233.... 252.-4+ 2253:. o: W 2.03.0. *uuumox u...“ ¢w>_._ AilMiOVOIOVU [I‘ll-I'll", Figure 11. 73 Radiochromatography of the products of heart galactokinase reactions performed at 30 mM galactose and the effect of alkaline phos- phatase and mild acid hydrolysis on the iso- lated products. Galactokinase assays for heart were performed at 30 mM D-galactose [2 uCi] as outlined in Figure 6, with a final volume of 0.1 ml. The radioactive products were isolated by paper chromatography, treated as shown on the Figure and rechromatographed. Details of chromato- graphy, enzymatic, and acid hydrolyses are des- cribed in the Materials and Methods section. The vertical arrows denote the regions of the chromatogram corresponding to hexose and hex- ose monophosphates. RADIOACTIVITY 74 HE ART ‘HEXOSE-P’ ORIGIN I No TrsotmsM +Alkollns Phosphate s +Mlld Acid Hydrolysis A L A s l s A 1 L 1 j 1 ‘HEXOSE‘ CM FROM ORIGIN l l 4.; 5 IO I5 Figure 12. 75 Radiochromatography of the products of brain galactokinase reactions performed at 30 mM galactose and the effect of alkaline phos- phatase and mild acid hydrolysis on the iso- lated products. Galactokinase assays for-brain were performed at 30 mM D-galactose [2 uCi] as outlined in Figure 6, with a final volume of 0.1 ml. The radioactive products were isolated by paper chromatography, treated as shown on the Figure and rechromatographed. Details of chromato- graphy, enzymatic, and acid hydrolyses are des- cribed in the Materials and Methods section. The vertical arrows denote the regions of the chromatogram corresponding to hexose and hex- ose monophosphates. RADIOACTIVITY 76 BRAIN "uexosE-P ‘HEXOSE; ORIGIN No Trsoimsni +Alkollns Phosphatase +Mild Acid Hydrolysis L n 4 s 1 J 1 4 s l n J L A l s l n l O 5 IO IS 20 25 CM FROM ORIGIN 77 Figure 13. Hexokinase activities and distributions in rat brain, heart and liver. Activity is measured in nmoles glucose phospho- rylated/min/gm fresh weight tissue. Values are the averages of triplicate determinations on 2 animals i S. D. 78 w- eanoaom m >.:>:.U< mmm may owe mosHm> one .cwououm mfi\cmE\+owuo¢ .ommcmm nonpasop oumnmmonmim omoxmn Hops: moonumz was mamauoumz cw confluomop who mponuofi wanna 110 o o o va.m mansaom o o o ev.~ HmEomouowz o o o e~.N Hmwumcosoowmm samum xowao o o o m¢.¢ manuaom o o o o HmEOmonon o o o Hm.o Hmwupcogoouwz ammo: you c mm.o o om.mm mansaom o o o mm.m HMEOmOHOflz o o o ev.HH Hmwupconoouwz samum ppm 0 mm.o o oa.m mansaom mh.o hm.m mm.o mm.v HmSOmouowz o mm.~ vm.o vm.m Hmwupsonoouwz Hmbfln pom +de +mnfiq pom aw mowufl>fluo¢ mmmcomouphnoo mumnmmonmio omouomamu can mumsmmonmlm omoosaw unopcomooi+adz was +moflz mo sofiusnauumwo HoHsHHoonom .m manna Figure 14. 111 Gas chromatographic separation of galactose 6- phosphate and glucose 6-phosphate standards and chick brain extracts. Trimethylsilyl derivatives of standard galactose 6-phosphate (peaks 1-4) and glucose 6-phosphate (peaks 5 and 6)[lower trace] and chick brain extracts [upper trace] on 1% OV-l [4 ft.) at 200°C. The mass spectra presented in Figure 15 were taken from the corresponding standard B-D— galactopyranosyl 6-phosphate and chick brain extract (peaks 4). mmmmmmmm mmmmmmmm 113 .v xmmm no va ousmwm cw pousomoumou who mobwum>wuop one .>o on as oopnooou ouo3 Huon H030H. pomuuxo swung xowno Scum o>fium>flnmp mcwpcommouuoo map was Huoam Hommsg mumnmmonmlm Hamocmnmmou Iowammiolm pumocmum mo mo>wum>fluoo Hmawmanumawuu on» no swuommm mum: .muozmmonmim Humane“ Ihmouomammiolm pumvcmum can cache xoflno mo muuoomm moms o>flumnmmfioo .ma musawm 1.14 80mm 088m8mOmVOO¢DmMDOmOmNOONOQOOF Om DDDD>I>!IF w v— NNO Nmm me o\E oi BM mum umn mmN 850mm8m0mm800m¢8v0mm8m .OmNOONOme 00.. On ’IDDDDDIDDDDI‘DDDDDI DDDDDDDDDD «\E mvN EN ..mv VON n3. mN_ mu O—x m—m hon hmm mmN ij mVN EN v x08 8928 c165 onO 5. B} mNe VON nhll! o\oV\uw o -..ooe Nome: was: oloeooosooéoogoos otooeoa 115 .mponuoz new mamfiuoumz cw confluomop mm weapon luom oum3 mNMmmm one .cflouonm mE\cHE\pouwpflxo mloiamw moHOEc :H co>flm mm muflooHo> .mcoflumuucoocoo ousammonmlm mmouomamm msflmum> cues mua>wuom ommcomoupanmp oucnmmonmim mmouomamm 0Hn9H0m olllb Gamma can olllo HmEOmouoflE Ho>HH How uon huwooam>loumuunndn m .moauw>wuom ommcomoupmnop oumgmmosmim omouomHmm unoccmmool+mndz mandaom :Hmun new one HmEomouowE Hm>wa umn mo somwummeoo owuocwx « .ma ousmwm 116 assess _v amp—.355”— n N _I+ w T # ALIDO'IEIA Figure 17. 117 Gas-liquid chromatographic separation of the tri- methylsilylated products of rat liver microsomal and rat brain soluble NADP+-dependent galactose 6-phosphate dehydrogenase activities. The reaction mixtures were prepared for gas-1i- quid chromatography as described in Materials and Methods. The separation of the reactants and products prior to the incubation period, with and without alkaline phosphatase treat- ment, are shown in tracings A and C, respec- tively. Tracings B and D show the separation of the reactants and products after the in- cubation period, with and without alkaline phosphatase treatment. Peaks 1, 2, and 3 are 7, a, and B-D-galactose (30), respectively. Peak 4 is an unknown and peak 5 is D-galactonate. DETECTOR RESPONSE 118 TIME (mM.) 3 "var brain 3 2 1 i 4 \ 3 A | 2 A 3 i 3 1 3 s 4 B 4 A C ‘ x D 2 6 I0 14 Figure 18. 119 Lineweaver-Burk plots of rat brain of galactose 6-phosphate (H. 0—0) , and galactose 1- phosphate (0—-—0) phosphatase activities. Assay methods are described under Materials and Methods. Velocities are in nmoles galactose released/min/gm fresh tissue. 120 0.6 F as, " ' 6P 1 0.4 '- V _ - 6P 0 0.3 '- O.2 - ' 1P 0.1 -. . ' 1’] .. . . ; ; . [GALACTOSE-P (mM)] , Figure 19. 121 Rat brain 6-phosphogalactonate phosphatase acti- vity as a function of 6-phosphoga1actonate con- centration. The assay method is described under Materials and Methods. The reaction velocity is in nmoles phosphate released/min/gm fresh tissue. VELOCITY 122 l l I l I I 2 ' 3 4 5 ESP-GA LACTONATE «mM] Figure 20. 123 Effect of galactose 6-phosphate and 6-phospho- galactonate on rat brain phospho-glucose isomerase. A substrate-velocity plot of brain phospho-glucose isomerase activity (O—-O) in the presence of ga- lactose 6-phosphate (0.2 mM, 0--0 and 1.0 mM, t—-—t) and 6-phosphogalactonate (0.4 mM,C1-{J). Velocity is given in pmoles fructose 6-phosphate formed from glucose 6-phosphate/min/gm fresh tissue. VELOCITY 3 n O 124 0.5 1.0 [GLUCOSE-6P (mM)] 125 Figure 21. Effect of galactose 6-phosphate and 6-phosphoga- lactonate on rat brain glucose 6-phosphate dehydrogenase. A substrate-velocity plot of brain glucose 6- phosphate dehydrogenase activity (0--O) in the presence of galactose 6—phosphate (0.2 mM, O--O and 1.0 mM, H) and 6-phosphoga1actonate (0.4 mM, s—-—i). Velocity is given in pmoles glucose 6-phosphate oxidized/min/gm fresh tissue. VELOCITY 126 0.8- 0.7 - O.6 .. 005 '- o,4 .- 0.3 0.2 O 0.5 [gLUCOSE-op (mMfl 127 Figure 22. Effect of galactose 6-phosphate on rat brain sol- uble hexokinase. A substrate velocity plot of soluble brain hexo- kinase (0-—-1) in the presence of galactose 6e phosphate (0.2 mM, t—-—i and 1.0 mM,:3-t3). Velocity is given in pmoles glucose phosphoryl- ated/min/gm fresh tissue. '06 1.4 'o VE160CITY in .0 o 0.4 0.2 128 1 l l 1 OJ 0.2 0.3 0.4 [GLUCOSE (111 Mi] 0.5 Figure 23. 129 The effect of 6-phosphogalactonate on rat brain 6- phosphogluconate dehydrogenase. A Lineweaver-Burk plot of 6-phosphogluconate dehy- drogenase activity in the presence of increasing amounts of 6-phosphoga1actonate. Assay methods are described in Materials and Methods. Velocities are given in pmoles 6-phosphogluconate oxidized/ min/gm fresh tissue. IO 130 [op-GLUCONATE (mM 3 y. I- ’V 9’6 6 «‘3‘ ' K? g i- G“. :5 °" 91“ O . Oo A o.‘ “‘ I . NO 59'6“. " .. s I"! b l 4 l l 1 1 i, s 1 O IO 20 I GO ‘0 SO 131 .osmmflu ammum Eb\CHE\poNHUon mumcoodamonmmozmlm moHoE: cw co>wm mum mowuwooHo> .mponuoz can mamwuoumz Hopes omnwuomop ohm moonuos mound .ommcmmouphnmp oumcooaamonmmonmim sauna pom pom oumcouomHmmosmmonmim mo coaumsmeumuop AM may now uon coxwa .vm madman 132 wees: mem .ofiom owuoomouoHsowuu woa ca oumnmmonmum omouomHmm pom oumnmmosmaa mmouomamm mo mwmhflouomm .mm onsmflm 145 L 1 1 I 1 1 I 1 1 1 1 1 1 V: e. '0 .— ... c’ “souvauosav 20 15 I0 HYDROLYSIS TIM E IminJ 146 .xwocommm on» ca conauomoo ouo mGOHufiocoo Houcoawuomxm .AOIIIOV ocouooaocouooaom oco ouocouomHom mo Eon on» “fielllev ouozmmonm owcoouocw "ucomoumou maonfihm .ouocouooaomonm Imogene oouoouu omouozmmonm ocflaoxao Bonn oomooaou ouonmmonm oasomuocw poo ocouooaocouooaom oso ouocouooaom no son on» coo3uon muuofiownoaoum .om ohsoem 147 11d- ?Es m2: 2295.52— 00« co. 0 fiduddiqd‘JTdedddqldqflqfifid- O 100 no.0 [W09 ISH'IOW '1' "I‘a‘ Figure 27. 148 Gas liquid chromatography of 6-phosphogalactonate and alkaline phosphatase treated 6-phosphogalac- tonate. In Frame A peaks represent: (1) a-methyl-mannoside [standard]: (2) galactono-y-lactone; (3) galacto- nate. In Frame B peaks are: (l) a-methyl-manno- side [standard]; (4) 6-phosphogalactonic acid. Experimental conditions are described in the Appendix. #‘n‘ 149 i 3 l ... l in Z . mate 2 as m Ill 8 '1': 3 nM' 1-1 2 [I , U Ill .— w Lx a ‘ A: A i 4 I! 1 J 6 l0 :4 IS 22 26 so 34 as 42 is so TIME (min.) 150 .xflocomm4 one CH oonwuomoo ouo mcowuwocoo Houcoefluomxm .ouonmmonmim omocoummouooammnolm one 6 Ave poo Amy poo ouosmmoom no omoqousmouooammuanm can a Ame poo AHO upcomoumop m oEoum ca mxoom .ouosouooaomonmmonmlm “HO upcomoumou m oEoum :H mxoom .ocouooaocoosam Ionmmonmim Ame oco ouocoosHmosmmonmlm Adv “ucomoumou H oEon :H mxoom .ouonmmonmiw omouooaom poo oumcouooaomonmmonmim souocoooamonmmonmnw mo mnmoumouofiouso owswwa moo .mm ousmfim 151 5.5231: s v— e— e he.mmOhUwuoommoH omocoummouooaomlolm one a Ame poo Ave .omocousmouooaomioio Ame "ohm mxoom U ofioum cH .ouocou Iooaom Ame poo osouomairlocouooaom AHV "usomoumou mxoom m com 4 moEoum nH .omouooaom poo ocouomaocouooaom .ouocouooaom mo mnmoumouofiouno owswwa mow .mm ousmflm 153 BSNOdSII 30133130 14 10 l4 1 TIME (min.) l4 “MIMI!“IIWIWHIHIH 3 0337