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AI ‘_ ‘i ‘ - 7| 4" -.)‘ . ‘ v“. .1 ‘2 3‘ 1 . fiV’ w I t (3/ . f \. 11"1',‘ DW- c—g,- h~~--.-.._ / M This is to certify that the dissertation entitled PHOSPHORYLATION 0F LYSOSOMAL MEMBRANE COMPONENTS presented by Christine Ann Collins has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry Major professor Date €//7/§3 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 RETURNING MATERIALS: )V1531_J Place in book drop to LIBRAxlgs remove this checkout from .‘ll-r‘nlsl. your record. FINES will V be charged if book is returned after the date stamped below. ROOM USE ON]. ’ DO NOT CIRCU TE PHOSPHORYLATION OF LYSOSOMAL MEMBRANE COMPONENTS By Christine Ann Collins A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1983 I37-2vs/‘0 ABSTRACT PHOSPHORYLATION OF LYSOSOMAL MEMBRANE COMPONENTS By Christine Ann Collins In order to examine whether regulation of lysosomal function could be mediated by modification of membrane components through phosphoryla- tion, lysosomal membranes were treated with [t-32PJATP and the labeled products were characterized. Two phosphorylated components were detected. One of these was found to contain 32P in an acyl linkage. This phosphorylated product exhibited rapid turnover and sensitivity to pH and hydroxylamine characteristic of other acylphOSphorylated enzymes, most of which are cation pump ATPases. Acylphosphate formation occurred in the absence of a divalent metal cation, but the rate and extent of phosphorylation were increased in the presence of M92+. Calcium did not stimulate 32P incorporation. However, dephosphorylation was stimulated by either Ca2+ or MgZ+. Acylphosphate fonnation was decreased by treatment of the membrane with inhibitors of the lysosomal membrane ATPase, such as N,N'-dicyclohexylcarbodiimide and sulfhydryl reagents. Polyacrylamide gel electrophoresis and autoradiography demonstrated a labeled band, approximately 180,000 daltons, exhibiting properties of the acylphosphate moiety. Phosphoryl transfer activities similar to those found for other acylphosphorylated ATPases were detected in lysosome Christine Ann Collins preparations. These results are consistent with the identification of the acylphosphate as a covalent reaction intennediate of a lysosomal membrane ATPase. The second phosphorylated material was identified as phosphatidyl- inositol 4-phosphate and a trace of phosphatidylinositol 4,5-bisphos- phate based on its chromatographic properties on silicic acid. Chromatographic and electrOphoretic analysis of the deacylated lipids further substantiated this conclusion. The enzyme which carried out the phosphorylation reaction, phosphatidylinositol kinase, was characterized with respect to assay conditions required for optimal activity and the use of exogenous phOSphatidylinositol as a substrate. The specific activity of the kinase in lysosomes was comparable to the activity found in liver microsomes and plasma membrane, the previously recognized sources of this enzyme. Stimulation of phosphoinositide metabolism has been observed in many tissues in response to hormones and other agents which modify calcium flux in the cell. It is possible that some of the recognized effects of honnones on liver lysosome function may be mediated through regulation of polyphosphoinositide turnover in the lysosomal membrane. To My Parents 11' ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. William Wells for his support and guidance throughout the course of my graduate studies. I would like to thank the members of the lab, particularly Charles Smith and Jeffrey Nickerson, for helpful discussions, both scientific and otherwise. I would like to acknowledge the many friends I have made during my graduate career, especially Mary Tierney. Their encouragement helped me to get through the rough periods when nothing seemed to work. Finally, I would like to thank my husband, Russell Kohnken, for his love and confidence in me. TABLE OF CONTENTS page LIST OF TABLES ...................................................... vii LIST OF FIGURES ..................................................... viii ABBREVIATIONS ....................................................... ix INTRODUCTION...... ........... ... .............. .. .............. . ..... 1 LITERATURE REVIEW The vacuolar apparatus of the cell ........... . ........... ..... 3 Receptor-mediated endocytosis ................................. 3 Lysosome isolation methods....................... ........ ..... 4 Characterization of the lysosomal membrane .................... 5 Acidification of the lysosomal compartment.................... 8 Hormonal regulation of lysosomal activity ............. . ....... ll Membrane fusion............................... ........ ........ ll Calcium and polyphosphoinositide metabolism ................... 12 REFERENCES .......................................................... l5 CHAPTER I. CHARACTERIZATION OF ENDOGENOUS PROTEIN PHOSPHORYLATION IN ISOLATED RAT LIVER LYSOSOMES .................. ..... ....... 23 Abstract .................................................. 24 Introduction......... ....... ........ ...... . ....... . ..... .. 24 Experimental Procedures Materials ..... ......... ................................ 24 Membrane preparation.... ............................... 24 Standard phosphorylation assay .............. . .......... 25 Polyacrylamide gel electrOphoresis........ ............. 25 Other assays ...... . ........... . ........................ 25 Results Time course of membrane phosphorylation................ 25 Polyacrylamide gel electrophoresis of labeled products. 25 Effect of cyclic nucleotides on phosphorylation........ 26 Substrate specificity of the phosphorylating activity.. 26 Chemical characterization of the phosphorylated products.................................. .......... 26 II. III. Discussion ................................................ ReferencesoooooOOOOOOOOOOOOOOOOOOOOOOO00000000000000. 00000 IDENTIFICATION OF PHOSPHATIDYLINOSITOL KINASE IN RAT LIVER LYSOSOMAL MEMBRANES. O O O O O O O O O O 0 O 0 0 O O O 0 0 O O O O O O O O I O O O 0000000 Abstract. ................................................. Introduction ........... . .............................. .... Experimental Procedures Materials........... ..... . ...... . ................. ..... Lysosome preparation......... ..... ..................... Lysosome phOSphorylation............................... Extraction of lipids................................... Chromatography of lipids ................... ............ Chromatography of deacylated products .................. Phosphatidylinositol kinase determination .............. Protein determination ........... . ...................... Results Identification of 32P- labeled phosphatidylinositol 4— —phosphate.................. ..... ......... ....... Pr0perties of lysosomal phosphatidylinositol kinase Assay conditions ............................... ..... Effect of Triton X-100... ........................... Effect of phOSphatidylinositol ............ ' .......... Time course of phosphatidylinositol 4- -ph05phate production. ...... ...... ... ..................... PI kinase in nonnal rat liver lysosomes ............. Subcellular localization of PI kinase. .. ....... ..... Inhibition of PI kinase................ ..... . ....... Discussion ............ ............ ....................... . References ........ . ........ . ........... . .................. CHARACTERIZATION OF AN ACYLPHOSPHATE INTERMEDIATE OF A LYSOSOMAL MEMBRANE ATPase....... .......................... Abstract..... ............................................. Introduction........ ........ ...... ...... .................. Experimental Procedures Materials.... .......................................... Lysosome preparation ...... ..... ........................ Phosphorylation assay....... .......... ‘ ............... .. Polyacrylamide gel electrophoresis. ................ ATPase assays ..... ..... . ........ ............. . ...... . NTP- NDP phosphate exchange....... ............. ......... 27 28 29 3O 30 31 32 32 32 32 33 33 33 33 34 35 36 37 ATP-P- exchange.... .................................... 42 Prote1n determination .................................. 43 Results Substrate specificity of the acylphosphorylation reaction.. ............... ..... .................. 44 Polyacrylamide gel analysis of the acylphosphate.. . 44 Cation requirement for acylphosphorylation... ....... 50 Cation requirement for dephosphorylation ............ 50 Substrate specificity of the lysosomal membrane ATPase .......................................... 55 Effect of inhibitors on acylphosphate formation and ATPase activity ................................. . 55 Phosphoryltransfer reactions ..... . .................. 58 Discussion ................................................ 64 References .................................... . ........... 74 SUMMARY ............................................................. 77 vi LIST OF TABLES Table Page Literature Review I. Lipid Composition of the Lysosomal and Plasma Membranes... 6 II. Carbohydrate Composition of the Lysosomal and Plasma Membranes ............................................... 7 Chapter I I. Effect of Cyclic Nucleotides on Membrane Phosphorylation.. 26 II. Substrate Specificity of Lysosomal Phosphorylation Activity ................................................ 26 III. Characterization of Reaction Products ..................... 27 Chapter II 1. Activity of PI Kinase from Lysosomes, Plasma Membrane, and Microsomes .......................................... 33 II. Activity of Lysosomal PI Kinase in the Presence of Inhibitors ..... . ........................................ 33 Chapter III I. Acylphosphate Formation in Lysosomal Membranes ............ 49 II. Substrate Specificity of Lysosomal Membrane ATPase ........ 56 III. Effect of ATPase Inhibitors on ATPase Activity and Acylphosphate Formation... ...... .... ......... . ..... ..... 57 IV. Effect of Sulfhydryl Reagents on ATPase Activity and Acylphosphate Formation... ..... . ...... ...... ............ 6l V. Phosphoryl Transfer Reactions of the Lysosomal Membrane... 63 VI. Summary of ATPase Partial Reactions under Selected Experimental Conditions ....... . ......................... 70 Figure Chapter 1. Chapter I. 2. 3. Chapter 1. 2. 3. LIST OF FIGURES Page I Time Course of Endogenous Phosphorylation in Rat Liver Lysosomal Membranes .................................... . 25 Time Course of Endogenous Phosphorylation ................. 25 TDAB-Acid Gel Electrophoresis of Phosphorylated Lysosomal Membranes ..................................... 26 Factors Affecting Lysosomal Membrane Phosphorylation ...... 26 Effect of pH on the Rate of Hydrolysis of Phosphorylated Products ............................................... . 27 II Analysis of the 32P-labeled Material from Lysosomes ....... 31 Assay Conditions for Lysosomal PI Kinase .................. 32 Effect of Triton-X 100 on Lysosomal Phosphatidylinositol Kinase .................................................. 32 Effect of Phosphatidylinositol Concentration on PI Kinase Activity ......... . ...................................... 32 Time Course of the Phosphatidylinositol Kinase Reaction... 32 Time Course of DPI Formation in Normal Liver Lysosomes.... 32 III Nucleotide Specificity of Phosphorylation Reaction ........ 45 Analysis of Acylphosphate on LDS Polyacrylamide Gels ...... 47 Metal Ion Specificity for Phosphorylation. ...... . ......... 5l Metal Ion Requirement for Dephosphorylation ............... 53 Effect of pH on Vanadate Inhibition of ATPase ............. 59 viii ABBREVIATIONS Abbreviations not listed below are standard usage according to the Instructions to Authors, 1983, in the Journal of Biological Chemistry. ATPKS adenosine 5'-[X—thio]triphosphate CaM calmodulin CDTA trans-cyclohexane-l,2-diamine-N,N,N',N'-tetraacetic acid DCCD N,N'-dicyclohexylcarbodiimide DPI phosphatidylinositol 4-phosphate EGTA ethylene glycol bis(A-aminoethyl ether) N,N,N',N'-tetra- acetic acid FITC fluorescein isothiocyanate LDS lithium dodecylsulfate MOPS 4-morpholino-propane sulfonic acid NEM N-ethylmaleimide NDP nucleoside diphosphate NTP nucleoside triphosphate pCMB p-chloromercuribenzoic acid PI phosphatidylinositol SDS sodium dodecylsulfate TDAB tetradecyltrimethyl ammonium bromide TPI phosphatidylinositol 4,5-bisphosphate 2-ME 2-mercaptoethanol ix INTRODUCTION Hormones, particularly insulin and glucagon, are known to have effects on proteolysis in liver cells (1). Glucagon greatly stimulates cellular autOphagy, proteolysis, and lysosomal membrane swelling and fragility (2). In the fasting state, increased degradation of intracellular proteins by the lysosomes provides a pool of amino acids. Insulin and exogenous amino acids counteract this effect. Thus, glucagon and insulin control intracellular protein catabolism, which may be a major regulatory point in hepatic gluconeogenesis. The studies to be described here were undertaken with the objective of determining whether phosphorylation of lysosomal membrane components occurs. The effects of glucagon on cellular metabolism are in most part carried out through a phosphorylation-dephosphorylation mechanism modulated by cyclic AMP (3). Insulin also affects the phosphorylation of soluble and membrane proteins, including its own receptor (4-6). Zahlten gt_al. reported that glucagon stimulated the net uptake of 32P1, in vivo, into rat liver proteins of microsomes, mitochondria, and lysosomes (7). An initial study of lysosomal phosphorylation, in vitro, also found stimulation of 32P labeling by cyclic AMP (8). This effect was later found to be due to contamination of the crude lysosomes with soluble or other organelle-associated protein kinases. The phosphorylation of more purified lysosomal membranes has been examined and the results of these studies are presented here. 2 Chapter I was published in the Journal of Biological Chemistry, volume 257, pp 827-831 (1982). Chapter II was published in the same journal, volume 258, pp 2130-2134 (1983). This work is reprinted here by permission of the publishers. Chapter III was written in a format suitable for publication in the same journal. LITERATURE REVIEW The vacuolar apparatus of the cell. Lysosomes (“lytic particles“) were first identified as a membrane-bound, or latent, form of acid phosphatase (9). Lysosomes are now known to contain over 60 acid hydrolases, most of which are glyc0proteins (10). This organelle is unique in that its size is quite variable and the lysosomal contents are heterogeneous. In fact, the word lysosome refers to a variety of organelles, termed the vacuolar apparatus (11), which are involved in the digestion of every class of biological material from endogenous and exogenous sources. Among these organelles, primary lysosomes are those whose enzymes have not engaged in a digestive event. Secondary lysosomes are sites of present or past digestion. Receptor-mediated endocytosis. Secondary lysosomes may arise from fusion of primary lysosomes with vesicles derived from the plasma membrane. These vesicles have been referred to as endosomes, endocytic vesicles, and receptosomes (12, 13). The process of endocytosis and subsequent transfer of material to the lysosomal compartment has been examined in some detail for such exogenous ligands as low-density lipoprotein, transferrin, asialoglyc0proteins, peptide hormones, bacterial toxins, viruses, and lysosomal enzymes themselves (12, 14). The initial fonnation of an endocytic vesicle relies on the binding of these ligands to a plasma membrane receptor, invagination of this area of the membrane (coated pit), and then release of the coated vesicle into the cytOplasm. The clathrin coat is rapidly Shed and the smooth 3 4 vesicle may then fuse with another endosome or with a lysosome. Lysosomes may in some cases also phagocytize endogenous material (15). A portion of cytosol or internal membrane structure becomes segregated by a limiting membrane of as yet unknown origin. This phagosome fuses with the lysosomal membrane to form an autOphagolyso- some. These secondary lysosomes may be involved with many digestive events until undigested material builds up within the lysosomal membrane. The structure is then referred to as a residual body, con- taining electron dense material, membrane remnants, and a highly pigmented material, lipofuscin (9). This also occurs in pathological states of lysososmal storage disease, where the lack of a Specific hydro- lase leads to a build up of unmetabolized material in the lysosome (16). Lysosome isolation methods. It is very difficult to purify liver lysosomes away from other cellular organelles. Differential centrifu- gation schemes do not result in adequate separation of lysosomes from mitochondria and peroxisomes. The method currently in wide use involves injection of rats with the detergent Triton NR-1339 (17). This material is taken up by the liver by adsorptive pinocytosis and is accumulated in the lysosomes where it cannot be degraded. The secondary lysosomes containing this material have a lowered density and can therefore be separated from contaminating cellular components with a relatively high yield. The disadvantages of this method are that only secondary lysosomes are obtained, i.e., those lysosomes which have (fused with endosomes carrying the detergent, and the possible alteration in membrane structure and enzymatic activity caused by this material (17-20). Other purification methods include: 1, uptake of dextran-SOO by lysosomes with a corresponding increase in density (21); 2, density gradient centrifugation in metrizamide (22); and 3, free 5 flow electrophoresis (19). These methods also result in a relatively high degree of purification. However, these methods also probably select for only secondary lysosomes. Percoll density gradient centrifugation (23) has been utilized to separate two p0pulations of lysosomes, probably corresponding to primary and secondary lysosomes (24). The yield and degree of purification are not as high as for the other methods, however. Characterization of the lysosomal membrane. In order to carry out cellular hydrolytic functions, the limiting membrane of the lysosome must fuse with that of the vesicle carrying the substrate. Therefore, the regulation of lysosomal activity in the cell may well occur at the level of membrane interactions. In order to examine lysosomal membrane function, several studies of the lipid (19, 25-30), protein (25, 30-35), and carbohydrate (25, 31, 36) composition of the membrane have been carried out. The lipid composition (Table 1) is quite similar to that of the plasma membrane. These membranes and the Golgi apparatus are characterized by large amounts of cholesterol and sphingomyelin. In addition, the fatty acids found in lysosome and plasma membrane lipids show a high degree of saturation (25, 37). The lysosomal membrane has a unique lipid, bis(monoacylglyceryl)phosphate, which is synthesized from lySOphosphatidylglycerol and an acyl donor, probably phosphatidyl- inositol, on the lysosomal membrane (38). The similarity between the lysosomal and plasma membranes has led some researchers to suggest that large amounts of plasma membrane are incorporated into secondary lysosomes (19, 29). However, the carbohydrate contents of the lysosomal membrane (Table II) is higher than that of the plasma membrane, and the protein and glycoprotein composition is entirely different from that of other subcellular .Aom .mmv mum; umymmcu1:0pvch soc; mcowpmcwawca mcmgnams mammpa .emcwscmpmn uoza $.m .Amm .NN .apv m85¢momap umppwc-coS.Leu.u.a .Ampv mwmmcocaocuompw zopwnmmcw x5 vmgwamca mmsomomzbw 0.0 N.m umewacmuwcz 1 N o.m m.m_ m.m N.¢ mumzamosahchmuzmexumocosvmwn - N o._ - m.~ o.e eva._o.ecau P.mm _N m.o~ o._N «.mm 8.“ =._a>som=.;am m.© w m.e m.m m.e N.m FoumeCEPAcwpagamoga N.m m_ N.P m.m _.m o.m acwcmmpscapagamoga .u.: .c.: m.u.= P._ N.F m.N mewsmpocmgum_»u_pa;amoga0ma_ w.o_ o_ F.e_ N.op N.m_ m.©~ peasapocacuapxcwuagamosa m._ e e.m w.~ N._ m.o mewpogu_»o_paaamo;a0m»_ c_.me mom ue.am do.mm N.©m we._e m=__o;u.»uwpa;amo;a mzocoggmoga cwawF we & ocmgnsmz Mammy; mcmcnsmz Fmsomong cwawg .mwcmgnswz mammpm vcm meOmOng ms» mo cowuwmoqeou uwawb .H wpnmp Table II. Carbohydrate Composition of the Lysosomal and Plasma Membranes. Carbohydrate Lysosomal Membrane Plasma Membrane pg/mg protein neutral sugars 45.63 135.1b 197C 28.0a glucosamines 25.4 14 trace 37.0 galactosamines 5.5 25 8.6 sialic acid 16.1 52.1 28 10.4 aLysosomes and plasma membranes from Triton-treated rats (25). bTriton-filled lysosomes (31). cNormal lysosomes (36). 8 organelles (25, 35). There do not appear to be any enzymatic activities common to both the lysosomal membrane and the plasmalemma (33, 34). In addition, the rate of turnover of lysosomal membrane proteins and carbo- hydrate residues is different from that of plasma membrane (35). This suggests that either the region of the plasma membrane which invagin- ates to fonn endocytic vesicles is of a much different composition than other areas of the membrane, or that the components of the endosome do not remain with the lysosome but are recycled back to the cell surface. Evidence has been accumulating in support of the second hypothesis (12, 14, 34, 39-41). Plasma membrane markers are constantly recycled between surface and internal compartments. The half-life of cell surface receptors is much longer than would be calculated based on the rate of endocytosis and degradation of the receptor-associated ligand. Hence, there must be extensive reutilization of endosone membrane components and little incorporation of these into the lysosome. Acidification of the lysosomal compartment. The majority of lysosomal hydrolases have a pH optimum in the acid range (37). The pH of the lysosome in living cells has been measured by the uptake of a pH-sensitive fluorescent dye (42), and determined to be in the range of 3-5. Maintenance of the acid pH was dependent on energy, since the addition of metabolic inhibitors led to an increase in lysosomal pH and a resulting inability of the lysosomes to carry out hydrolytic functions. The presence of an energy-dependent proton pump has been postulated by Mego (43), based on the increased proteolytic activity of isolated lysosomes incubated at pH 8 when ATP was added to the medium. No stimulation by ATP was observed at pH 5, suggesting that the lysosomal proteases were already Optimally active at this pH. Studies measuring the ATP-dependent uptake of basic dyes (44), methylamine (45), 9 and amino acid methyl esters (46) into lysosomes have also led to the conclusion that a proton pump is active in the lysosomal membrane. The uptake of these substances is based on their diffusion across the membrane in an uncharged state and subsequent protonation in an acidic compartment. The compounds in the ionized fonn are then unable to cross the membrane. This mechanism may also hold true for the anti- inflammatory drug, chloroquine, which is concentrated over 1000-fold in the lysosomes compared with the cytosol (47). This compound, other "lysosonotropic" amines, and NH4Cl inhibit a number of lysosomal functions, such as protein degradation (48-50), receptor recycling (40, 51, 52), and fusion of the lysosome with endosomes and phagosomes (53-56). The inhibitory effect of the amines is believed to be due to an elevation of the lysosomal pH. Continuous uptake of these compounds as observed in cultured cells would therefore require constant adjustment of the pH by means of an energy requiring proton pump (58). A second mechanism for maintaining low pH is a Donnan equilibrium established by negatively charged glyc0proteins within the lysosome. This mechanism probably accounts for the difference of 1 pH unit found between isolated lysosomes and their external medium (59), although it is not responsible for formation of the initial acid conditions (60, 61). Direct evidence for the existence of a proton pump in lysosomal membranes has been obtained by Ohkuma gt_gl. (57, 62). They examined the fluorescence of a dextran-dye conjugate which had been sequestered in secondary lysosomes. The fluorescence of the isolated organelles was proportional to the pH of the internal compartment. ATP was found to decrease the fluorescence due to the lowered pH within the lysosomes. Various metabolic inhibitors and ion0phores were found to inhibit this activity, further substantiating the existence of an ATP-driven proton 10 pump in the lysosomal membrane. An ATPase activity in the lysosomal membrane has been analyzed by Schneider and others (46, 63-65). It was suggested that this enzyme carries out the translocation of protons across the membrane (45). In the studies described in Chapter III, various inhibitors of known proton pump ATPases were found to also inhibit the lysosomal enzyme. Since this activity is non-latent and exhibits a neutral pH optimum, it has been suggested that the active site of the enzyme is located on the cytOplasmic face of the lysosomal membrane (64). An ATP-driven proton pump in the membrane of adrenal medulla chromaffin granules has been well characterized (67-69). The physiol- ogical function of the acid pH in these granules is to promote the accumulation of the basic catecholamines against a concentration gradient. This proton pump exhibits sensitivity to ionophores and metabolic inhibitors and has been characterized as having an electro- genic mechanism. An ATPase activity has been measured in chromaffin granule membranes which is thought to be responsible for acidification (70, 71). The chromaffin granule and lysosomes may be related in both structure and function since they are both derived from the Golgi (39), and lysosomal hydrolases are secreted from the adrenal cell along with the catecholamines. A similar acidification activity has also been observed in the sperm acrosome, which is a modified lysosome at the head of the sperm which fuses with the plasma membrane in the capacitation reaction (74, 75). An energy-dependent proton pump has also been postulated for the membranes of yeast vacuoles and secretory vesicles from many sources (76-83). The possible role lysosomes may play in fusion of these vesicles with the plasma membrane and subsequent secretion has been reviewed (72, 73, 84-86). Proton pump 11 activity has also been found in endosome membranes (87, 88). The acid pH within this vesicle promotes dissociation of ligands from their receptor. The receptor may then be recycled back to the cell surface and the ligand may diffuse into the cytoplasm or be delivered to the lysosome for degradation (12). Hormonal regulation of lysosome activity. Most studies of lysosomal function involve the measurement of proteolysis under various honnonal (89) and nutritional (90, 91) conditions. Glucagon has been shown to enhance proteolysis in the perfused liver and in isolated hepatocytes (92-94), while insulin and excess amino acids inhibit breakdown. It was proposed that the glucagon effect is mediated by a decrease in intracellular amino acids, particularly glutamine (95). Ogawa gt_gl. have examined the perfused liver microsc0pically and have found that glucagon or cyclic AMP in the perfusate stimulates the formation of autophagolysosomes (96, 97). Lysosomes in these electron micrographs were seen to elongate and wrap around intracellular structures such as mitochondria. This suggests that the lysosomal membrane may be altered in some way by glucagon treatment. Glucocorti- coids have also been found to stimulate proteolysis in isolated hepatocytes (98). It has been postulated that this occurs independ- ently of glucagon and insulin effects, perhaps by stimulation of membrane protein synthesis required for autophagy. These experiments suggest that the regulation of lysosome fusion with other membranes may be the important factor in modulating lysosomal hydrolytic activity. Membrane fusion. Studies of lysosome fusion with plasma membrane and phagosomes have been carried out in vivo and in vitro (66, 99-103). A recent report has demonstrated the fusion of isolated endocytic vesicles with lysosomes (104). Vesicles which retained their clathrin 12 coats were unable to fuse. Uncoated endososmes, and those stripped of their surface proteins by protease treatment were capable of fusion, suggesting that lipid components are of primary importance in this process. Fusion events cannot be indescriminant, however, since lysosomes only fuse with newly formed endocytic vesicles, only rarely with older vacuoles, and never directly with mitochondria or the nucleus (105). Secondary lysosomes may fuse several times with endosomes, phagosomes, or other secondary lysosomes, however. It has been suggested that the similarity of the plasma membrane and the lysosomal membrane allows recognition and fusion of these elements (30, 105). However, as discussed before, the protein and carbohydrate composition of these membranes is quite different. Again, it may be that the lipid components, which are quite similar for these membranes but different from other cellular structures, are the important recognition point. The high concentration of sphingomyelin, cholesterol, and saturated fatty acids in these two membrane systems confers imperme- ability and a large degree of structural rigidity (25). The only unique lipid in the lysosomal membrane, bis(monoacylglyceryl)phosphate, arises from interaction of the lysosome with other membranes which contain phosphatidylglycerol or cardiolipin. Secondary lysosomes, e.g. Triton-filled lysosomes, have been found to contain much higher levels of this lipid than primary lysosomes (27). The effect of this lipid on membrane structure and function, however, is unknown. Calcium and polyphosphoinositide metabolism. Calcium is known to play a role in membrane fusion, both from studies with artificial lipid bilayers (106, 107) and in studies of secretion, where secretory vesicles fuse with plasma membrane during exocytosis (108-110). Extra- cellular calcium is required for the release of both catecholamines and 13 lysosomal enzymes from the adrenal gland (111). Calciun is also required for the release of acid hydrolases from the polymorphonuclear leukocyte (66). Some investigators have suggested a role for a Ca2+- dependent ATPase on the membrane of fusion competent vesicles (66, 108, 112). ATP stimulates fusion of secretory vesicles with plasma membrane (112, 113), but the mechanism of this action is not completely understood. The class of myo-inositol containing phospholipids has been proposed to play a role in membrane fusion in secretory cells (66, 114-116), myoblasts (117), erythrocytes (118), and lysosome fusion in leukocytes (116). The polyphosphoinositides, phophatidylinositol 4- phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI), exhibit rapid turnover, particularly in response to honnones and other agents which stimulate calcium flux in their target cells (119, 120). Phosphatidylinositol kinase has been found in liver plasma membrane (121), microsomes (122, 123), nuclear envelOpe (124), and in adrenal chromaffin granule membranes. The data to be presented in Chapter II indicate a lysosomal membrane localization for this enzyme as well. The further phosphorylation of DPI to TPI is catalyzed by a particulate enzyme in erythrocytes (126) and kidney (127), and by a soluble enzyme in brain (128). The degradation of the polyphosphoinositides has been extensively studied with regard to soluble and membrane bound phospho- monoesterases (122, 129), as well as soluble and membrane bound Ca2+- dependent phosphodiesterases (130-132). The latter enzyme may be stimulated by hormones binding to their cell surface receptor and the subsequent increase in cytosolic calcium levels (133-135). The reaction product, 1,2-diacylglycerol, may reenter the biosynthetic scheme through phOSphatidic acid, cytidine diphosphoglyceride, and back 14 to phosphatidylinositol. The inositolphosphates also produced may be further degraded by phosphomonoesterases (129). The enzymology of this phosphoinositide turnover phenomenon has been recently reviewed (136). The phosphatidylinositol cycle is thought to occur largely on the plasma membrane. However, the presence of polyphosphoinositides on the lysososmal membrane leaves Open the possibility that this pool of lipid is also hormonally controlled (137). Besides their possible function in calcium translocation and membrane fusion, polyphosphoinositides have been implicated in the propagation of nerve action potentials, plasma membrane ion and solute transport, and the orientation and modulation of enzymes (119). Phos- phoinositides have been found to serve as anchorage sites for several plasma membrane-associated enzymes (138). A requirement for phospha- tidylinositol for the maintenance of membranous ATPase activities has also been found (139, 140). 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(1980) Biochem. J. 192: 783-791. Kirk, C.J., Michell, R.H., and Hems, D.A. (1981) Biochem. J. 194: 155-165. Prpié, V., Blackmore, P.F., and Exton, J.H. (1982) J. Biol. Chem. 257: 11323-11331. Irvine, R.F. (1982) Cell Calcium 3: 295-309. Wells, W.W., and Collins, C.A. (1983) in Lysosomes in Biology and Pathology (Sly, W.S., and Dean, R.T., eds.) vol. 7, (in press) North-Holland, Amsterdam. 22 138. Low, M.G., and Finean, J.B. (1978) Biochim. Biophys. Acta 508: 565-570. 139. Mandersloot, J.G., Roelofsen, B., and Gier, D.E. Jr. (1978) Biochim. Biophys. Acta 508: 478-485. 140. Lipsky, J.J., and Lietman, P.S. (1980) Antimicrob. Agents Chemother. 18: 532-535. 141. Hendrickson, H.S., and Reinertsen, J.L. (1971) Biochem. Biophys. Res. Commun. 44: 1258-1264. CHAPTER I CHARACTERIZATION OF ENDOGENOUS PROTEIN PHOSPHORYLATION IN ISOLATED RAT LIVER LYSOSOMES 23 lll .otauu. or BIOLOGICAL L .«uisrn \ol '5? Vol.1.“ 0! Januan ls pp 337-“. . 190.12 Pmuedul L'. 5.4 24 Characterization of Endogenous Protein Phosphorylation in Isolated Rat Liver Lysosomes* Christine A. Collins and William W. Wells; (Received for publication. June 22. 19611 From the Department othochemwtry. Michigan State University East Lansing, Michigan 48824 Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphoryla- tion activities. The transfer of phosphate to membrane fractions from [yr-”HAT? was analyzed by gel electro- phoresis under acidic denaturing conditions. Two phos- phopeptides were detected. with molecular weights of 3.000 and 14.000. Phosphorylation of these proteins was unaffected by the addition of cAMP. cGMP. or the heat- stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP- dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14.000-dalton ”P-labeled product was formed rapidly in the presence of low concentrations (250 an) of either Ca“ or Mg”. This product was labile under both acidic and alkaline conditions. suggesting that this protein contains an acyl phosphate. present presumably as a catalytic intermediate in a phospho- transferase reaction. The lower molecular wei ht species required a high concentration (5 mat) of M ‘ for phosphorylation. and micromolar concentrations of Ca" stimulated the high-dependent activity. The addition of Ca" and cal- modulin stimulated the phosphorylation reaction to a greater extent than with Ca” alone. This activity was strongly inhibited by 0.2 mas LaCl; and to a lesser extent by 50 pm chlorpromazine or trifluoperazine. These re- sults suggest that the 3000-dalton peptide may be phos- phorylated by a Ca” -'-'—“' '-- J " ‘ kinase as- sociated with the lysosomal membrane. Many membrane systems have been studied which show reversible phosphorylation of membrane-associated proteins (11. In most cases. the presence of membrane-bound protein kinases as well as the endogenous protein substrates has been demonstrated. While membrane phosphorylation may be sen- sitive to hormonal regulation. via cAMP or other effectors. a change in function or activity upon phosphorylation has yet to be demonsuated for the majority of these prOteins. How- ever. the phosphorylation-dephosphorylation of membrane proteins could provide a mechanism for the alteration of membrane structure and regulation of transport and enzy- matic scrivities (2). In 1972. Zahlten et aL 131 showed that glucagon stimulated ' This work was supported by Grant AM10209 from the United States Public Health Service. It was presented in part at the 72nd Annual Meeting of the Amencan Society of Biological Chemists In St. Louis. MO. June 119811. Fed Proc. 40. 1661, The costs of publi- cation of thll article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertise- ment" in accordance with 15 15.5.0 Section 1734 solely to indicate this fact. 3 To whom inquiries should be addressed the incorporation of ”P. into membrane proteins of rat liver microsomes. mitochondria. and lysosomes in two. This result suggests that phosphorylation of lysosomal membrane pro- teins may occur by a cAMP-dependent process. Lysosome phosphorylation may. therefore. mediate some of the known effects of glucagon on hepatic lysosome function. such as increased proteolysis in the nutritionally deprived rat (4. 51 and formation of flattened vesicles and nut- LW‘ 3 “ in livers perfused with glucagon or cAMP 16.71. We previously reported the tn vitro phosphorylation of rat liver lysosomes isolated by Percoll density gradient centrifu- gation 18). Phosphorylation of lysosomes isolated from 3-day starved rats was stimulated by CAMP. whereas the activity in lysosomes from fed rats was unafieCted or inhibited by the same concentration of cyclic nucleotide. In the present report. we exartune the phosphorylation of more highly purified liver lysosomal membranes prepared from rats injected with the detergent Triton WR- 1339. wERIMENTAL PROCEDURES Motermls—[”PlOnhophosphate. carrier tree. was purchased from New England Nuclear. [y- “PlATP was prepared by the method of Glynn and Chappell 191. as modified by Reunann et at. 1101. Calmod~ ulin was purified to homogeneity from frozen bull testis according to the procedure of Dedman et a1. 1111. Materials for gel electrophoresis were purchased from Bio-Rad. Tmluoperaune was prov1ded by Snuth. Kline. and French Laboratones. Chlorpromazine. tetradecyl- tnmethyl ammonium bromide iTDABI.‘ bovine heart protem lunase inhibitor. type I CAMP-dependent protein kinase. cyclic nucleotides. and protein kinase substrates were obtained from Sigma. Tnton WR- 1339 was from the Ruger C hermcal Co. All Other chemicals for enzyme assays were of reagent grade. Membrane Preparation—Triton WR-1339-filled lysosomes were prepared from rat liver by the method of Leighton et al. :12). Holtzman strain rats weighing between 150 and 250 g were mjected intrapemoneally with Triton Wit-1339 185 mg/100 g body weight» .3 days before sacrifice. Lysosomes were isolated by differential centrif- ugation and flotation thrOugh 34.5% twm-I sucrose in the presence of phenyunethylsulfonyl fluoride :75 mg/literi and soybean trypsm 1n- hibitor c 100 mg, liter; Lysosomes obtained from the sucrose gradient were washed by diluting wi .h an equal volume of cold 20 mat Tris- HCl pH? 5. After centrifugation at 40. (I!) x g for 20 min. the pellets were suspended in 10 mat Tris-HC 1. pH 7.5. containing 1 mst dithio- threitol. This lysosome preparation was purified 60-fold over the whole homogenate based on heaoaamimdase specific activity 1131. Contamination of the preparation by mitochondria. Golgi. and per- onsomes was negligible based on {umarase l 141. galactosyltransferase (151. and urate oxidase 416) actmnes. respecnvely Analysis of SAD” cytochrome c reuuctase «17) indicated less than 5‘? contammauon by endoplasmic reticulum. Analysts of 5'-nucleotldase I151 or alkaline phosphodiesterase 1191 indicated less than 49 contamination of the lysosomes with plasma membrane. Membranes were prepared by 1 cycle of freeze-thawing and collected by centrifugation at 80.000 x g for 45 min. The resulting pellet was resuspended in 10 mat Tris-RC1. ‘The abbreviations used are TDAB. tetradecyltrimethyl ammo- nium bromide: CaM. calmodulin 25 Lysosomal Membrane Protein Kinase pH 7.5. and 1 ms dithiothreitol and stored at -70 'C. Membranes prepared in this manner typially contained 35% of the protein present tnthe intact lysosome fraction. Microsomes were isolated from rat liver as described in Ref. 20 and were purified 3-fold over the whole homogenate based on glucose 6- phosphataae and NADH cytochrome c reductme activity. Plasma membrane. isolated according to the method of Emmelot et 01. (21). was purified 25-fold based an alkaline phosphodiesteraae activity. Standard Phosphorylation Assay—The reaction mixture con- tained lysosomal membranes (SO-1(1) ug of protein). 50 ml Tris-RC1. pH 7.5. 5 mar MgCla. and 0.1 ml (y-”P]ATP (ND-200 cpm/pmol) in a final volume of 60 iii The reaction was initiated by the addition of membrane sample and incubation at 3!) ‘C was carried out for various times as indicated. The reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid containing 10 mar Na pyrophoaphate and 20 ul of bovine serum albumin (5 mg/ml). After 10 min on ice. the samples were filtered through Whatman 3MM filter paper discs and washed with 10 ml of 10‘? trichloroacetic acid. The filters were dried and counted in a Beckman model 7000 liquid scintillation spectrom- eter. For gel electrophoresis. the reactions were stopped with 0.5 ml of 10% trichloroacetic acid containing 2 mm ATP. After 5 min on ice. the aammee were centrifuged at 12.000 X g for 5 min. The precipitated proteins were washed by centrifugation with 0.5 ml of 10% trichloro- acetic acid. 2 mat ATP. and with 0.5 ml of 50 mar KHIPOe/HIPOa. pH 2.0. The pellet was solubilized in 50 iii of 0.25 is sucrose. 2‘5 2- mercaptoethanol. 35 nus TDAB. 100 mu KHzPO. (pH 4.0). and 10 ug/ml of methyl green. Polyacrylanude Gel Electrophoresis—Cationic detergent acid slab gels containing 10% (w/v) acrylamide were prepared according to Amory et at. (22). The gel solution contained 0.09% (w/v) TDAB and 75 mm KHzPO./lLPO.. pH 2.0. The 4% stacking gel solution was prepared with 125 mu KHIPOA. pH 4.0. The method of Jordan and Raymond (23) was used for polymerization. The electrode bufler containing 75 max glycine and 0.125% lw/v) TDAB adjusted to pH 3.0 with HsPO..Thegelwasrunatconstantcurrentofe0mAforthat 4 'C. After electrophoresis. the gels were soaked for 5 min in 2‘1 glycerol. dried. and exposed to xcray film for autoradiography. Molecular weights of phosphoproteins were estimated by comparison with pres- tained molecular weight standards obtained from Bethesda Research Laboratories To quantitate the radioactivity in some cases. protein bands were cut from the dried geL rehydrated in 0.5 ml of 8:0 for 30 min. and counted in 5 ml of scintillation mixture. Other Assays—Fromm content was determined by the method of lowry (24) using bovine serum albumin as a Calmodulin content was determined by the procedure of Sharma and Wang (25). RESULTS Time Course of .Vembrane Phosphorylation—The incu- bation of lysosomal membranes with [y-"PlATP resulted in a time- and concentration-dependent incorporation of radio. activity into trichloroacetic acid-insoluble material. The ex- tent of phosphorylation after 1 min of incubation increased linearly with the amount of membrane sample up to 2 mg of protein/ml (data not shown). A rapid incorporation of label was seen (Fig. 1A) which peaked at 30 s and then diminished. A second component was phosphorylated at a slower rate. This produCt was stable to treatment with hot trichloroacetic acid as indicated in Fig. 18. whereas the early peak disap- peared. The difference plot derived from these curves is shown in Fig. 2.4. indicating the time course of the acid-labile ”P- labeled producr. The data in Fig. 2B show that under condi- tions of low Ca’” or Mg’ concentration. an early burst of phosphorylation also occurs. A slow increase in ”P incorpo- ration is then seen in the presence of Ca" but not Mg‘. Polyacrylamide Gel Electrophoresis of Labeled Products—The time course and acid-alkali stability of the phosphorylated products were examined by TDAB-acid poly- acrylamide gel electrophoresis and autoradiography (Fig. 3). After 30 s of incubation. both a 3.000 and 14.000-dalton band were labeled. However. by '2 min of incubation. the “.000- dalton band disappeared. Treatment of the reaction mixture after 1 min of incubation with 10% trichloroacetic acid at [:Pt/‘/O/. i i L 2 3 4 3 S I If 1 -_.L..._.J J h N 0 —L . -4 l 3 (I) q. J T l ‘5 "e- mconeoasrtou ts moi/ml 0 N 0 1 .1 2 ‘3 3— 5 TIIIE (min ) Fic.1 Time course of endogenous phosphorylation in rat liver lysosomal malmembranas. A. incorporation of 1''1’ into total membrane proteins Assays were conducted as described under ‘Ex ' tal Procedures” for the times indicated. B. incorporation of into an acid-stable product. Samples phosphorylated as above were treated with 10% trichloroacetic acid at 90 °C for 20 min. After cooling the tubes on ice for 10 min. the protein precipitate was collected by filtration as described. Each point represents the mean a S. E. of 4 experiments 0 i ”r. mcoeeonsnou ts anal/mg) TlUElninl Pic. 2. Time course of endogenous phosphorylation. ..-1 in- corporation of ”P into acid- labile product. These values were obtained by difl’erence from the curses presented in Fig. 1. B. phosphory latinn under conditions of low metal ion concentration. Assays were carried out as usual except for the substitution of m gm Ca" 250 int Mg" (H) for the 5 mu Mg‘" in the assay mixture. Each point represents the mean of 2 experiments. A A 90 °C resulted in the loss of the 14.000-dalton product but only a slight decrease in the intensity of the 3.000-dutch band. Treatment with 1 st NaOI-I at 90 °C degraded bath phospho- rylated produCts. Other factors were tested for their ability to affect phospho- rylation of the lysosomal membrane (Fig. 4). In the presence of low metal ion concentration. only the 14.000-dalton band was present (lanes 0 and c). The 3.000-dalton band appeared , when 5 mas Mg” was included in the reacuon: 5 mM Ca" did not effectively replace Mg" (lane b). The phd‘sphorylation of the 14.000-dalton product was inhibited by these higher salt concentrations. The addition of Ca“ and CaM resulted tn increased phosphorylation of the lower molecular weight 26 Lysosomal Membrane Protein Kinase l4K—w Ill-- - - .- FIG. 3. TDAB-acid gel electrophoresis of phosphorylated ly- sosomal membranes. The time course and acid base stability of the phosphorylated products were examined Samples were treated as ducribed below and prepared for electrophoresis and autoradiogra- phy as described under "Experimental Procedures" The arrowheads indicate the position of the top of the stacking gel and the dye front. a-c. assays were conducted at 30 'C for the times indicated a. Ill-s incubation: b.1min;c.2mindande.Thesamplesweretrsatedas described after a Main reaction; :1. 20-min incubation with 10% trichloroacetic acid at so 'C; e. 10-min incubation with 1 N NaOH at 90 'C. l Nl-sp .- 3("' ‘- -.' s b c d e l g I Pic. 4. Factors aflecting lysosomal membrane phosphoryl- ation. All assays were carried out {or 1 min at 30 'C with additions to the reaction as indicated below. TDAB-gel electrophoresis and autoradiography were carried out as before. a. 250 int Ca” (no Mg“): b. 5 mar Ca" (no Mr“); C. 250 an Mr"; d and e. 5 ml Mg". Hi. 5 mar Mg". 0.8 ug of CaM. and 50 int Ca"; I. no further addition; 3. 50 ms chlorprornasine: h. 0.2 ml LsCli. product. This stimulation was blocked by 50 its! chlorproma- zine (lane 3). The phosphorylation of this band was com- pletely inhibited by the addition of 0.2 ms! LaCla (lane Ii). To test whether the phosphorylation observed was due to the presence of small amounts of other membranes in the lysosomal preparation. we examined the phosphorylation of purified microsomes and plasma membrane. Lysosomes. mi- crosome. and plasma membrane preparations were phosphoo rylated and subjected to TDABogel electrophoresis and au- toradiography. Phosphorylated products similar in molecular weight to those found in the lysosome sample were observed in both the microsome and plasma membrane samples. To quantitate J"P incorporation. these portions of the gel were cut out and counted. The radioactivity in the 3.000-dalton region of the gel was 2.2-fold higher in the lysosome sample than in the microsome and 9-fold higher than in plasma membrane with equal amounts of protein applied in each case. Multiple bands were apparent at molecular weights greater than 10.000 in the microsome and plasma membrane lanes. but the lysosome sample contained at least 2-fold more "P in the 14.000-dalton region of the gel. These data indicate that the phosphorylated products detected are of lysosomal origin and can not be explained by contamination with other membranes. Effect of Cyclic Nucleotides on Phosphorylation—The phosphorylation of lysosomal membranes was nor signifi- cantly aflected by the addition of cAMP or cGMP (Table I). cAMP-dependent kinase inhibitor also had no effect. The addition of cAMP and type I cAMPodependent protein kinase did not result in additional incorporation of radioactivity. These experiments were aLso carried out by analyzing the products of the reaction on TDAB-acid gels. No additional radioactivity was detected in either phosphopeptide. and no other phosphopmtein was labeled by exogenous cAMP-de- pendent protein kinase. Substrate Specificity of the Phosphorylating Activity—The ability of lysosomal membranes to phosphorylate exogenous substrates was examined (Table II). In expen'ment 1. the commonly used substrates of CAMP-dependent kinase were tested. In experiment 2. known substrates of Ca”-dependent protein kinase were examined. There was no increase in ”P incorporation in the presence of these substrates beyond the activity seen with the membrane sample alone. Analysis of the reaction products by autoradiography following gel elec- trophoresis revealed no incorporation of ”P into the exoge- nous proteins. Chemical Characterization of the Phosphorylated Prod- ucts—The products of the phosphorylation reaction after 30 s and 5 min of reaction were analyzed as described in Table TAIL: 1 Effect of cyclic nucleotides on membrane phosphorylation Lysosomal membranes were phosphorylated {or 5 min as described under “Experimental Procedures" with the indicated additions made to the assay bufler. Additions il"-incor'i:n:n'atiscl‘ penal/m protein None so: : {7.3 (1%) 5 int cGMP 97.1 t 15.0 (107) 5 ml CAMP 87.6 x 12.7 (97) 74.8 = 20.8 (82i 654 : 11.0 (72) 5n! cAMP+15ungln‘ 5 ins cAMP + 10 pg CAMP-dependent protein kinase ' Mean value z 5.51. of 4 experiments. Numbers in parentheses are per cent of control assay. ' CAMP-dependent protein kinase inhibitor. Tastz l1 Substrate specificity of lysosomal phosphorylation activity Phosphorylation amays were conducted for 5 min as described under “Experimental Procedures" with the addition of the indicated substrates. Added nib-Irate 'P-mcorporated‘ pistol/In; protein Experiment 1‘ None 152 (100i Casein 155 (102) Historic II-A 1.32 (8h Protamine 105 :69) Bovine serum albumin 10;: (68» Experiment 2‘ None its (1001 Phosphorylase b 108 (94) Myosin 74 (65i ' Mean of 2 determmations. Numbers in parentheses are per cent of control assay containing no additional substrate. . '250 ug of the indicated protein were added in a final reaction Volume of 100 ul "Assays were conducted in the presence of 1“) ms Ca" and 100 pg of the indicated substrate in a volume of 60 pl. 27 Lysosomal Membrane Protein Kinase III. The data indicate that the product formed at 30 s is an acyl phosphate. The material was unstable in hot acid and alkali. The 12% of radioactivity remaining after hot trichlo- roacetic acid treatment is due to the small amount of the phosphate ester formed by 30 s. The phosphate ester com- prised 95"? of the total phosphorylated product at 5 min of incubation. Neither product was solubilized by extraction with organic solvents. indicating that phospholipids do not make up a significant portion of the J‘P-labeled material. Trypsin TABLE III Characterization of reaction products Lysosomal membranes were phosphorylated for the times indicated below as described under "Experimental Procedures." The nP-labeletl products were mixed with 100 pg of bovine serum albumin and precipitated with 10‘! trichloroacetic acid and 10 mar Na pyrophoa- phate and collected by centrifugation at 12.000 x g for 5 min. The samples were treated as described below and the remaining radioac- tivity determined by collecting the trichloroacetic acid-insoluble ma- terial on paper filters as before. 1) Control samples were resuspended in 1 ml of cold 10% trichloroacetic acid. 2) Samples suspended as for control were heated at 90 °C for 20 rrun and then chilled on ice. 3) Samples solubilised in 0.2 ml of 1 N NaOH were heated at 90 °C for 10 min and precipitated with 1 ml of cold 10% trichloroacetic acid. 4) Samples were washed in 1 ml of cold distilled water and extracted 3 times with 1 ml of chloroform/methanol (2:1. v/vl. 5) Samples were treated as in 4 and then extracted 3 times with 0.5 ml of chloroform/ methanol containing 025% HCl. 61 Samples precipitated without serum albumm were solubiliued in 0.2 ml of 0.25 It Tris-HCl. pH 7.5. and treated with 10 pg of protease/ 100 pg of lysosomal protein at 30 ‘C for l h. 1 ml of cold trichloroacetic acid was added and the precipitated protein collected as above. The control for this treatment consisted of incubation of the sample in the absence of protease. R I‘ . 'W . . , 'heannam so a 5 min 1: 1. Control 1m 1w 2 Hot trichloroacetic acid 11.9 94.6 3. Hot NaOH 0 0 4. Chloroform/methanol 91.5 90.6 5. Chloroform/methanol/HCl 89.1 84.7 6. Trypsin 44.3 51.8 ‘ Results are the mean of 3 separate experiments. r O ./O \ ./ ‘ 0:2 4 6 8 (O (2 pH Frc. 5. Efl'ect of pH on the rate of hydrolysis of phosphoryl- ated product. Lysosomal membrane samples were phosphorylated in the presence of 250 pm Ca“ for 30 s at 30 °C. The reactions were stopped by the addition of 1 ml of 10% trichloroacetic acid containing 10 mas PP. and 20 pl of bovine serum albumin (5 mg/mli as usual. The protein was collected by centrifugation at 12.000 x g for 5 min. The pellets were washed with cold distilled water. Each membrane sample was suspended in 0.2 ml of solution at the indicated pH (HCl. 0.2 is acetate. citrate. Tris-Cl. or bicarbonate; and incubated for 30 min at 30 °C. After addition of 1 ml of 10‘? trichloroacetic acid. the membrane samples were collected by centrifugation as before. The sediment was dissolved in 0.1 ml of 1 is Tris—HCl. pH 7.5. for scintillation counting. 6 J 8 3 N U ’ 2 P - Release (% ol total Inc or povohonl o treatment released approximately one-half of the radioactivity compared with that found in a control reaction incubated under the same conditions in the absence of added protease. However. the incubated control value for the 30-s product was decreased by 80‘! compared with unincubated controL indi- cating hydrolysis of the acyl phosphate under these condi- tions. The hydrolysis characteristics of the 30-s phosphorylated product formed in the presence of 250 pM Caz" were deter- mined as shown in Fig 5. The JarP-labeled material was most stable at pH 1. with rapid hydrolysis occurring at both pH extremes. The hydrolysis rate of the acyl phosphate in 0.1 M acetate buffer. pH 3.5. at 40 °C was determined (26). The rate constant was calculated to be 0.012 : 0.002 min" and the half-life of the acyl phosphate under these conditions was 56 min. This value is in close agreement with the rate constant observed for the acyl phosphate intermediate of the (Na'. K‘l-ATPase (26). Similar results were observed for hydrolysis rate and pH effects using the usual assay conditions (5 ms: Mg” and l-min incubation) and correcting for the amount of stable phosphorylated product remaining. DISCUSSION We have shown in this report that a membrane fraction derived from Triton WR-lmfilled lysosomes contained 2 distinct phosphorylation systems. The time course of phos- phorylation was biphasic. with a rapidly labeled component which decreased with time and a second more slowly phos- phorylated product. The rapidly labeled component was sensitive to treatment with hot trichloroacetic acid and was the major product formed under low Ca" or Mg” concentrations. It was nOt extracted by organic solvents ruling out the presence of la- beled phospholipids. The phosphoprotein contained an acyl phosphate by the following criteria. 1) It was not detected in standard sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue in acidic methanol‘; 2) the ”P label was removed by treatment with hot trichloroacetic acid or NaOl-i: and 3) it showed a pH profile and rate constant of hydrolysis which is distinctive and comparable with that observed for other proteins containing an acyl phosphate linkage. Analysis of the phosphorylated products by cationic detergent-poly- acrylamide gel electrophoresis at pH 2 revealed phosphopep- tides of M. - 3.000 and 14.000. Based on the time course of its appearance and its susceptibility to hydrolysis. we conclude that the ld.0(X)-dalton band contains the acyl phosphate moi- ety. Acyl phosphates have been isolated as catalytic interme- diates (in (Na'. K’l-ATPase (26. 27), (Cai‘. Mf’1-ATPase (28). and other phosphotransferases (291. Work in this labo- ratory and others has shown the presence of ATPase activ- ity(s) in the lysosomal membrane (30-32). It is. therefore. likely that the acyl phosphate observed here is the covalent intennediate of the catalytic subunit of an ATPase or other phosphohydrolase associated with the lysosomal membrane. The second component was also protein in nature and appears to contain a more stable phosphate ester. This peptide was presumably the substrate for a protein kinase associated with the lysosomal membrane. We attempted to assay for kinase activity by adding exogenous substrates to lysosomal membranes (Table [1). So far we have been unable to detect stable phosphorylation of any protein other than the endog- enous 3.000-dalton peptide. Some of the added protein sub- strates led to an inhibition of the endogenous activity. perhaps by nonspecific binding to the membrane site where phospho- rylation normally occurs. The protein kinase may. therefore. be highly specific for this substrate or may require the correct ’ C. A. Collins and W. W. Wells. unpublished results. 28 Lysosomal Membrane Protein Kinase orientation and proximity of the substrate in the membrane. Various modulators of protein kinase activity were tested for their efi'ect on lysosomal membrane phosphorylation. In our highly purified preparations. neither cyclic nucleotides nor the CAMP-dependent kinase inhibitor had any effect on the endogenous phosphorylation. contrary to what we sug- gested previously (8). We feel that earlier preparations were contaminated with cAMP-dependent kinase from the cytosol or other membranes. The increase in lysosomal membrane phosphorylation in response to glucagon observed by Zahlten et a1. {3) may be due to contaminating phoephoproteins from mitochondria or other source in their preparation Alterna- tively. phosphorylation of lysosomes by loosely associated cAMP-dependent kinase may be an important means of reg- ulating lysosomal function in vitro. Interestingly. we found no additional phosphorylation of lysosomal membrane compo- nents by adding type 1 cAMP-dependent protein kinase and cAMP to the reaction mixture. Tsung and Weissman (33) have observed cAMPvindependent phosphorylation in a ly- sosome-rich fraction from human polymorphonuclear leuko~ cytes. They reported the presence of heat-labile inhibitor of soluble cAMPvdependent kinase in the leukocyte lysosome preparation and attributed the lack of cAMP binding and stimulation of kinase activity in various fractions to the aetion of this inhibitor. We have also observed inhibition of soluble CAMP-dependent kinase activity by purified rat liver lyso- somes using histone as substrate2 but have not determined what efl'ect this activity may have on our measurement of endogenous lysosomal membrane phosphorylation. Ca" stimulate a number of cAMP-independent protein kinases. most of which require the presence of the Ca" modulator protein. calmodulin. for activity (34). We reported a small stimulation of phosphorylation by 1 mar Ca” in our previous work (8). Here we examined a much lower Caz’ concentration and the effect of CaM and inhibitors on the Ca”-stimulsted activity. We found variable degrees of stim- ulation by Ca” and CaM between preparations. but 50 as! Ca" generally stimulated phosphorylation of the 3.000-dalmn peptide 2-fold. Addition of CaM caused a further stimulation of 60%. Since the lysosomal membranes are prepared in the absence of metal chelators. it is likely that there is Ca‘“ already present in the sample. In addition. we have found that lysosomal membranes stimulate activator-deficient cAMP phosphodiesterase in the standard assay for calmodulin.2 This suggests that Ca" and calmodulin already present in the lysosomal membrane stimulate the endogenous kinase. The addition of 0.2 mas LaCh. which is ltnown to block Ca" transport and competes for Ca” binding sites (35). completely inhibited the phosphorylation of the 3.000~dalton peptide in the presence or absence of added Ca” and CaM. In addition, 50 psi chlorpromazine or trifluoperazine. drugs which inhibit CaM binding (36). decreased the Cal'. CaM-dependent stim- ulation of phosphorylation. These data suggest that Ca"’ may regulate the phosphorylation of the 3.000-dalton peptide through the acrion of a Ca”. CaM»dependent protein kinase associated with the lysosomal membrane. The existence of this Ca"-regulated phosphopeptide in lysosomes may provide a mechaan for the mediation of hormonal effects on lyso- somal function. REFERENCES 1. 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M., Rolleston. P. S., and Murray. R. K. (1978) J. Biol. Chem. 253. 2033-2043 21. Emmelot. P.. Boa. C. J.. van Heaven. R. P- and van Blitterswijlt. W. J. (1974) Methods Enzymol. 81. 75-81 22. Amory. A.. Foury. P. and Gofleau. A. (1980) J. Biol. Chem. 255. 935- Z). 9353- . 23. Jordan. E. M.. and Raymond S. (1969) Anal. Biochem. 27. 205- 211 24. Lowry. O. H., Rosebrough. N. J.. Farr. A. L. and Randall. R. J. (1951) J. Biol. Chem. 193. 265-275 Sharma. R. K.. and Wang. J. H. (1979) Adv. Cyclic Nucleotide Res. 10. 187-198 Nagano. K.. Kansaswa. T., Misuno. N.. Tashima. Y- Naltao. T.. and Nakao. M. (1965) Biochem. Biophys. Res. Commun. 19. 759-764 27. Nishigalti. 1.. Chen. P. T. and Holtin. L E. (1974) J. Biol. Chem. 249. 4911-4916 Degani. C.. and Boyer. P. D. (1973) J. Biol. Chem. 348. 8222-8226 Suzuki. R. Fukiushi. K. and Takeds. Y. (1969) J. Biochem. 66. 767-774 lritani. N.. and Wells. W. W. (1974) Arch. Biochem. Biophys. 164. 357-866 31. Mego. J. L. Farb. R. M.. and Barnes. J. (1972) Biochem. J. 128. 763—769 25. 26. .8938 Schneider. D. L (1977) J. Membr. BioL 34. 247—261 Tsung. P.-K.. and Weinmann. G. (1973) Biochem. Biophys. Res. Commun. 51. 336-842 Schulman. H.. and Greengard. P. (1978) Proc. Natl. Acad. Sci. U. S. A. 75. 5432—5436 Schatrmann. H. J.. and Burgin. H. (1978) Ann. N. 1'. Acad. SCR. 307. 125—147 Weiss. 8.. and Levin. R. M. (1978) Adt'. Cyclic Nucleotide Res. 9. W CHAPTER II IDENTIFICATION OF PHOSPHATIDYLINOSITOL KINASE IN RAT LIVER LYSOSOMAL MEMBRANES 29 Tu lotsiuu. or Biotmrcu. Catalan toll.“ No sinus u Februan- ‘3 pp. Medial. SA. ‘l'IlLflJ-l. I914 30 Identification of Phosphatidylinositol Kinase in Rat Liver Lysosomal Membranes“ Christine A. Collins and William W. Wells: (Received for publication. August 10. 1982) From the Department of Biochemistry. Michigan State University. East Lansing, Michigan 48824-1319 Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate ”P from [yJ’PIATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phoe- phate based on its chromatographic behavior on Silica Gel H thin lay er plates as compared with standard :L r '-—;.... The deacylation product, glyceryl- L L y.__-- "“ phosphate. was comp with standards tn chromatographic and electrophoretic sys- tems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4.5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was ac- tive at neutral pH in the presence of 20 mM MgC13. The addition of 0.4% Triton x-ioo stimulated the enzyme activity toward endogenous substrate. and the highest activity was observed in the presence of detergent and l mu phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X400. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergentpstimulated activ- ity of liver microsomes and plasma membrane. the previously recognized sources of this enzyme in the liver cell. Primary lysosomes from livers of starved rats ( 1) and those from livers perfused with glucagon or CAMP l2-5) rapidly undergo striking structural transformations in the process of autophagy. ln attempts to understand the biochemical basis for this process. we have investigated the possibility that endogenous phosphorylation of highly purified rat liver lyso- some components may affect membrane strucrure and func- tion l6) In this preVious study only two low molecular weight phosphorylated species were detecred when membranes from Triton WEI-1339 filled lysosomes were incubated with [ruP] ATP One was an acyl phosphate-containing protein of 14 000 daltons. We have suggested that this polypeptide represents the catalytic intermediate of a phosphotransferase reaction. The second phosphorylated component migrated in the 3.000- dslton region of sodium dodecyl sulfate or catiomc detergent gels. Identification of this material was complicated by the copurification of small polypeptides with the nP-labeled prod. ' This work was supported by Grant .001le from the United States Public Health Service. It was presented in part at the 12th International Congress of Biochemistry at Perth. Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adter. memes!" in accordance with 18 USC. Section 1734 solely to indicate this fact. 3 To whom inquiries should be addressed. uct. These behaved as proteolipids in their solubility in chlo- rofonn and elution from Sephadex G- 1m and Lil-20 columns. We have now shown that the low molecular weight material contains predominantly DPIl and a trace of TPI. Previous analyses of lysosomal membrane phospholipids have not dem- onstrated the presence of these polyphosphoinositides (7-9r. Nevertheless, Michell (10) noted that DPI has been found in adrenal chromsfin granule membranes (11. 12). and both polyphosphoinositides were present in the plasma membrane fraction from rat liver (l3. 14). He. therefore. speculated that membranes of the Golgi complex and lysosomes which are functionally related to these other membranes. may normally contain the acidic ”1,; r ' mes. We now provide evidence for the presence of phosphatidylinositol 4-phos'phate and phosphatidylinositol kinase (EC 2.7.l.67) in rat liver ly- sosomes. MENTAL PROCIDURES Materials—(”Wham carrier free. was purchased from New England Nuclear. [y-‘PJATP was prepared by the method of Glynn and Chsppell 115). as modified by Reimsnn et at. (16). Phos- phatidylinoaitol and other phospholipid standards were obtained from Serdary Research laboratories Poly-phosphoinositide standards. the triasmide and t, ' L ‘ mac-m acid were obtained from Sigma. Silica Gel H and cellulose (Avicel) thin lay er plates were from Analtech. Glass distilled organic solvents were ob- tained from MCB. Triton WR- 1339 was from the Ruger Chemical Co. 'hiton X-lm was from Research Products International. An Amines A-27 anion exchange high pressure liquid chromatography column (250 x 4 mm) was obtained from Bio-Rad Lysosome Preparation—Triton Wit-1339 filled lysosomes were prepared from rat liver by the method of Leighton e! at. (l?) as previously reported (6). omitting the wash step after sucrose gradient centrifiigation. These lysosomes were purified 60-fold over whole homogenate based on .— u’u. actmn tIEL Analysis of NADH cytochrome c reductase I19) and alkaline phosphodiester- ase (20) indicated less than 5% contamination by either endoplasmic reticulum or plasma membrane. Contamination by other organelles was less than 1‘}. Rat liver lysosomes were also purified by metnzamide gradient centrifugation according to the procedure of Wattiaua et al. «21). starting with the light mitochondrial bastion of DeDuve er al. (22). They were purified 50-fold over whole homogenate based on heaos- aminidase-specific activity. Contamination by mitochondria. micro- somes. and plasma membrane. was 1. l5. and 15‘}. respectively. Plasma membrane and microsomes were prepared from rat liver as described previously .6). Lysosome Phosphorylation—To obtain material for identification of the reaction products 0.5 mg of lysosomal protein was incubated with 2 mt [r‘PlATR 30 ml MgCh. and so mat Tris-RC1. pH 7.5. in a final volume of 0.5 ml for 5 min at 30°C. The ”P-labeled material was extracted and analyzed by thin layer chromatography as de- scribed below, ' The abbreviations used are: DPI ldiphosphouiositidei. phosphati- dylinositol 4-phosphate: Pl. phosphatidylinositol; TPI ltrtphosphoi- noeitide). phosphatidylinositol 4.5-b'nphosphate. 31 Lysosomal PI Kinase Extraction of Lipids—The phosphorylated lysosomes were ex- tracted with 1.5 ml of chloroformnsthsnol (1:2. v/v) followed by 0.5 (33).Tbeextxactionmixnirewasmixedthoroughlysndcentrifiged (lMngorIOmmlinlz-mlheevywalledPyrextubes.‘l'belower phasewasremovedtoanothernrbeandtheupperlayerandinterface wceexuecudagamwithlmldchlorofom'l'hecombinedlower phaseswerewashedwith2mlolmsthsnomuHCll1:1.v/v).andtbe upperphsaswasdismrdedaftercentrifiigation'l'helowerphnewas raplllynsutraliaedbyatmendmgsmsll plasticcups(KonteeGlsas Co.lcontaining15.1NNlLOHabovetheexn-ectinarubbersealed annnapbereforSmin‘lhechloroformlsyerwmmporatedto dryns-underaatreamofnitrogenatas-w‘CJ'helipidr-iduewas d'nolvedinaamsllvolumdmullof L'- ‘ ‘L "4.0 (TS-35:2. v/V/Vl in preparation for thin layer chromatography. For someprepamdons.0.5to1.0mgof authentic phosphatidylinositoH- mwandphoqhafidylinoaitol 4.5-bisphoaphstewereaddedas W: ofLipida—Aliquotsofthelipidextractwere applisdtoSilicaGelchinlayaplatesmOXZOcmlpreviously heetadatllO‘Cforlhandchromewgraphsdwithappropriste phogholipid inl-propanol:4.3itNl-l.0l-lcontaining10mis ‘ L " r ‘ acid (6535 Will (24) After develop- mentchromatogramswueerpoeedtox-rayfilmforautorediognphy. Pu phomholipid detection. the chrome were either arrayed withthephoqihstenagentofflochneretollfllorwithfrflailfuric acidoontsiningOfiilisodiumdichromsteandheatedatMO'Cform mm. W; o/Dedcylued Products—Aliquots ofths phoe- phorylated lmid fraction were deacylated following the procedure outl'med by Katee (B). The water- soluble products were chromato- graphed on celluloeethinlayerplatesin 2r- rotor-mp (6::31. v/v/vl. m2passssofthesolventmixnne. the chromatogramwasexposedtox-rayfilmandthensprayedwiththe phosphate reagent ofBochneretal. (25l.0thsraliquotsoftbewster- mluhleproductswareelsctrophoressdoncelluloaeplateslmxm unlin0.luaodiinnoxslste.pl-l1.5.withapotantialofMVfor45 Inm'l'hedriedplatsswereexpoeedtox-reyfilmfollowed bytreet- ment with the phosphate reagent The deacylated products were asbjectsd to ion exchange high performance liquid chromatography miscdumn(250x4mmlofAminexA-27mtheformateformand tedwithaOJIammoniumformste 20muammonium beratebufl'er.pl'l9.5. ataflowrateofO.6ml/min. Fortheseparation Misha-show! lipidprectireoraagr-adientwasgeneratedwitbmmlofowliaandO'ls .Iammoniumformsteeachcombinsdwithmmatunmoniumborate. mlof2.4rlHCland0.5mlofchloroformasdescribedbySchacht- WWKM Determination—The amaymixture containedsomss'l‘n's-HCLpH 7.5 ”mm. 2ml[y«”P1ATP lm-lmcpm/pmol).andlyeoeomeaampleinafinalvolume of0.1ml Except where indicated otherwise. thefinalproteinconcentretion was lmg/ml.’l'hereactionmstartadbytheadditionofATP-Mg“after a5—minpreincubationoftheothercomponsnuatfl’C.Afier axtracdonoflipidsbythemethodofSchachttfilasdeacribedabove. thschloroformpbssewasdrisdunderNiandther-iduesuspended ‘ma-nsll volume ofchloroformnnethsnol (21. v/v). The lipids were eeparatedonSilicaGelchinlsyerplstesintheunidirectional2- solvent system of Hauser et at. (24). The "P-lsbeled lipids were v'mialiaedby autoradiography. sndPl. DPI andTPl standards were detectedbynainingwithla. Areasofthethinlsyerplstecontaining radioactivitywereauapadofi'intovialscontainingfluorforscintilla- tioncmmting lnsotneexperimsntsthscombinsdchloroformextracts were washed twice with methanol-1 N HCl (1:1. v/vl and counted directlytomeasure'Pincorporation. WWW—Prompwashtaminsdbythsmethodof lawryetolfl‘llwithbovineearumalbuminasthestsndard. arson-s [Won of'P-Iobeled P‘ 7‘ “J," Phosphate .‘,‘. The chloroform extract of I'l‘hlabeled lysosomes was mixed with carrier DPI and TPI and subjected to chrometoyaphy on Silica Gel H thin layer plates. The solvent system contained a calcium chelator. t, ‘ “ ‘ ‘?--~n'r acid. which facilitated the migration of the polyphosphoinositides from the origin (Fig. M). It is apparent that the major a’P-labeled component corresponds in mobility to standard phosphati- dylinoaitol 4-phosphate. A trace of radioactivity was detected inthe region of L *3- " "4.5-bisphosphate. This varied from 3 to 5% of the label incorporated into DPI. The deacylated water-soluble products of the phosphoryl- ated lysosomal preparation were compared with the deacyla- tion products of standard phoephatidic acid. phosphatidylino- sitol. DPI. and TPI in three difi'erent separation systems. Fig. olthsgl detivetivesoftbecosrespondingphoqiho- lBilluatratuthattheradioactivematerialcomigrateswith A B + C O l" o O O . J V 0 U C O C p Q Q / A >2 V \-/1'\_/‘ C PA Pl DPI TPI Lye GP GPI GPIP 8MP; Lys GP GPI GPlPGPlPZ Lys Pic. 1. Analysis of the "P-lsbeled material from lysosomes. A. thin layer chromatography of lysosomal lipid extract Lysosomes were pbmhorylated and extracted. and the extract wm subjected to chromatography on Silica Gel H as described under “Experimental Procedures." The positions of lipid standards are indicated as detected by phosphate spray reagent. lanes 1-4 contained standard phosphatidic acid {PA}; Pl; DPI; and TPl. Thelyeoeome extract in lane 5 also contained carrier DP! and TPI. The lost lane shows the position of the lysosomal aP-labeled Jproduct 'm an autoradiograph of lane 5. B. electrophoresis of lipid deacylation products. Aliquots of lysosomal «labeled lipid along with carrier DPI and TPI were subjected to deacylstion in mild alkali (261.11» water-soluble products were electrophoresed as described under “Experimental Procedures.‘ lanes 1-4 contained the deacylation woducts of lipid standards: glycerol phosphate (GP); arm's: L " ‘ ‘ lGPD; dycerylphomborylinoeitol «phosphate (GPIP); glut. '. ' ‘ ‘ 4. 5-bisphosphste lGPIPal larie 5 contained the 'P- labeled lysosome extract and lane 6 is an autoradiograph of the separated deacylstion products. C. thin layer chromatography of deacylation products. The deacylstion producu of standards and the 3"Pvlabeled lysosome extract as described for B were separated on a cellulose thin layer plate in 2-propanol115 s‘ NECK: H10 (6:3:1. v/v/v). The lipids were detected by the phosphate spray reagent and autoradiography as before. 32 LysosomaIPIKinase r A a ‘l -A ‘ g’di'I e’O’M. 1 m t ' / J SOP ./ 1 i' 5 -’ i / ‘ em- I . / « 5 . / 2w 3 3” 5 T 7 s 7 °( pH (W. .8 Pro. 2. AusyoondltionsforlysoeomalPlkinasmA. pI-iprofileofl’lkinuesctivity. Ausyswereconducted asducrfiedunder‘ Experimental tsl"Prooedures inthepseeenceoflmsrPlsndO.4%TritonX-lmsttheindicated pfiThefollowingbuflersweremedstsfinalconcenn-snonofwmss: sodiumcin-ste. Tr‘u-msleste. Tris-CI. giycine-NsOl‘L‘Ihenulltssrethemeanof4determinatione8.eflectofMg“conosntrationonPlkinaseactivity. Auayswereconductedfor2minst30'CinthepresenceoflmssPLOA‘b'hitonX-lw. andtheindicated oonoentrationofMgCh.lmuEGTAwuaddedtothenectionniaunetomumesctivitymthesbunceofthe utionTheresultssrethemesnondeterminations. giycerylphomhorylinoaitol 4-phoephate when subjected to electrophoresis on cellulose thin layer plates. Similarly. chro- matography in 2-propanoL' 15 a momma (6:3:1, v/v/v) on celluloeeplatssrevesledssinglespot comigrstingwiththe deacylation product of authentic DPI (Fig. 10). In agreement with thus analyles. anion exchange high [l-ure liquid chromswtnphy of the deacylated material resulted in a ma- jor peak of radioactivity that eluted with glycerylphosphor- ylinositol 4—phnephate as detected by phosphate analysis. Again. a minor peak of radioactivity was found to elute with the deacylation product of TPI (data not shown). Properties 0! Lysosomal Phosphatidylinositol Kinase Assay Conditions—Lysosomal PI kinase setivity was found tobeoptimalstneutralpflandinthepruenceofstlesst'lo mil MgCl: (Fig. 2). The K. for ATP determined in the pesence of 20 ml MgCl: was 0.13 mas. A concentration of2 ml! ATP was utilized for subsequent experiments. Street of Triton X ~100—The nonionic detergent Triton X- Im stimulated PI kinase activity at a concentration of 0.4% (w/v) (Fig. 3). Inhibition of DPI formation occurred at higher concentrations of detergent using either endogenous lipid or added PI. The addition of 0.5 mas Pl in the absence of detergent did not enhance DPI synthea'm. The efl’ect of Triton X- I“) was found to be dependent on the sample concentration in the suay mixture. At concentrations of lysosomal protein leuthsn l Ina/ml. Tritonx- lOOwasinhibitorystsllconcen- cations tested in the absence of exogenous PI (data not shown) Effect ofm PI was utilized usuhscateforlyaosomslPlkinasessshowninFig. 4. Maximum incorporation of ’P into DPI was observed with 1 met PI in the presence of 0.4% Triton X-100. These condition were also found to give optimal incorporation of label into DPI of plasma membrane and microeome preparations. In the presence of both detergent and 1 ml PI. the reaction rate was linear with respect to sample concentration in the auay mixture up to 2 mg of protein/ml (data not shown). The Course of?‘ " ‘4, " '“'4—Pho¢hate Produc- tion—Analyst of the ”P-labeled reaction products on Silica Gel H plates showed that the major species was DPI. The rate of 'P incorporation into DPI is shown in Fig. 5. Using endogenous lipid as the substrate. the reaction rate proceeded linearly for only a short time. after which a plateau was reached To ausu whether this was due to depletion ofATP in the reaction mixture. ATPase activity was measured under these auay conditiona By 10 min of incubation at 3) °C. 75% d’theA'I'Phadbeenhydrolyxsdtogive’P..IfmoreATP wusddsdafterlOminofnscdomansdditionalburetof‘P LAJJ’" ..‘pg snot enigma... "V i \ and "liq herei- " 7 7 -L. ‘L_ -1. _ — 1 T_—:——T-__il Matcher-nee y l I I ‘1: es .1 u to Yuma Pun.“ do .nv uni ”II-she's» ‘ w w ~\. ' _._l-.._..._. pawl DWI-Q ' 0.- . O 7 Y I j/ l? N — Y g—f—W—‘I—TB— Pio. 3(toplefl).fleetof1fitonX-lfloaiyeoeoualphoe- yfinneitolePIkinasesuayswereconductedfor2min ufl'Cuducribsdundsr ”Expernnmtall’roceduee" minglOOpg oflysoaomalproteinandtheindicatedconcentrsn‘onof'h-iton de. “tenet-non utilised sndogenoulipidssubet‘rste (H) «0.5 mlPIaddedtothestandardauaymirture(C—O). ha.4(topright)£flectol ,L“_“ 4"ooneantra- tinnonPlkinaaeaetivity. Aueyswereconductedfor’zminstw ’Cinthepruenceon4$TfitonX~lmandtheindicatedconcenm- tionofpboohatidylinositol ho 5(bonomlem 'I'imecoureeofthe L kinase reaction Ausys were conducted as described under “EsperimsntalProceduree"with100ugoflysosomalprotein H.Plkinasesctivityun'ng mhsuateMO—OPI kinaseactivityinthepruenceofOAS Triton X-lma—Aactivity uu'nglmsIPIassubstrateinthepreaenceofOASTritonX-lm. ho. 6(boaomnght).1‘lmeom-eeofDPlfor-mationinnormal heerlyeoeometT'hePIhnsaeausywucsrriedoutuu-ulbut withlysosomupleparedbymetriaamidepadientcsnmnonas thesouceofenaymsandnibetrate. atsfinslooncenn-stionofo.5mg ofpotein/ml. hue (on: L ...) u .A I imorporstionwasobeervefldats notebown).'l"hefsctthsta platesuwasreschedwouldmggestthatlittledegradstionof theproductDPLoccuned. InthepresenceofOA‘B'I‘ritonX-lm. thereactionrstewas stimulated 2-fold. However. after reaching a- peak of 1’I’ in- corporationat 10min.therewassstasdydecreaseinthe amountoflabeledDPLInthepr-esenoeofsnopdmalconcen- tntionofexogenomubflate.theacdvitywufurtherstim- ulatedandfollowedthereactioncotn'seobeervedinthepree- enceofdetergentalone. 33 Lysosomal PI Kinase PI Kinase in Normal Rat Liver Lysosomes—Lysosomes prepared by metrizamide gradient centrifugation were ana- lyzed for the presence off“:L “1. ‘ ‘ “‘ kinase activity (Fig. 6). The activity was similar to that observed in Triton- filled lysosomes; however. DPI synthesis occurred up to 30 min. ATPase activity was lower in this preparation. degrading only50‘ioftheATP byGOmin. Subcellular Localization of PI Kinaee— Plasrns membrane and microsome fractions were isolated and assayed for PI kinase activity under the same conditions as for Triton-filled lysosomes (Table 1). Using added PI as substrate. the specific aetivities of the kinase from all three preparations were simi- lar. However. the plasma membrane preparation was strongly inhibited by Triton XolOO in the absence of exogenous sub- mm. Inhibition of PI Kinase—CaCla and LaCl. were tested for their eflect on P1 kinase activity (Table II. A). The stimulation observed in the presence of EGTA may be due to the chelation of inhibitory metal ions other than Ca“. since Caz’ at low Taste 1 Activity of?! kinase from lysosomes. plasma membrane. and microsomes Plkinaseauayswerecarriedoutforland2minatm°Cas described under “Experimental Procedures." The inin’al rate of reac- tion was determined in the presence of added detergent and substrate as indicated with protein at l mg/rnl for each sample. DPI Pormeuon' Additions Lysosomes Pm“ Miaosomes mailman/mg protein None 0.79 1.46 0.63 0.4% Triton X-lm 1.40 0.48 1.31 2.38 2.07 1.98 0.4% Triton X-100 +1 met P1 ' The results are the mean of 2-4 determinations Trust: 11 Activity of lysosomal PI kinase in the presence of inhibitors PIltineeeasseyswerecarriedoutfor2minst30'Cafiera5-min preincubation with all assay components except ATP and MgCli. The DPI fonned was extracted as described under “Experimental Proce- dures." The results are expressed as a per cent of the control incu- bation which contained 0.4% Triton X-100 and 1 mat Pl. Additions DITI formsu‘on‘ 't A. None 1m EGTA. 1 mil 111 COClr 10 nit 107 100 ms 105 1 mit 55 Lst 20 LEM 79 m int 22 B. Phosphatidic acid 0.2 mar 96 0.5 met 71 Phosphatidylserine 0.2 mM 98 0.5 mm 85 Phosphatidylcholine 0.2 mu 114 0.5 mm 124 Phosphatidylinositol 4-phosphate 02 inst 105 0.5 mar 93 ° The results are the mean of 2 experiments. The control P1 kinase specific activity is 2.33 nmol of DPI/min/mg of protem. concentrations led to a small increase in DPI formation At 1 mar Ca”. inhibition of DPI synthesis was observed. La” had been found to inhibit lysosomal membrane phosphorylation (6). Here it was examined for its effect of P1 kinase activity. At a concentration of 0.2 mM. DPI synthesis was inhibited 78%. The ability of other phospholipids to inhibit PI kinase activity was examined (Table II. B). In the presence of 1 mM PI. 0.5 mat phosphatidic acid was slightly inhibitory. A slight but reproducible increase in activity in the presence of phos- phatidylcholine was observed. Other phospholipids had little effect. DISCUSSION The presence of ‘L ‘ --‘- ' nl kinase in Triton-filled lysosomes and the formation of phosphatidylinositol 4-phos- phate. in vitro. establishes the occurrence of polyphosphoi» nositides in lysosomal membranes. PreVious reports of lyso- somal membrane phospholipid composition did not identify the polyphosphoinositides. presumably due to the very small levels present and their lebility under most emotion condi- tions. Using a"Pvlabeled ATP. it was possible to detect the synthesis of DPI in vitro and study the properties of the membraneoassociated phosphatidylinositol kinase. Recently. Behar-Bannelier and Murray (28) have reported that mouse liver microeomal membrane fractions incorporated significant amounts of ”P from [y-“PMTP into phosphati- dylinositol 4-phosphate. Their experience was similar to ours (6), that is. the observance of a fast moving band on polyacryl- amide gels of trichloroacetic acid-precipitated membranes following phosphorylation. In our studies. measurement of lysosomal membrane protein kinase activity gave erroneously high results by the trichloroacetic acid precrpitetion-filtration assay, since much of this activity is now known to result from lysosomal :“7‘ id," m“ ‘ kinase as reported herein. Consequently. we agree with the warning of Behar-Bannelier and Murray (28) that the formation of the polyphosphoinosi- tides in vitro may lead to an appreciable overestimation of the amount of ”P incorporated into endogenous membrane proteins. A difficulty in the detection of PI kinase activity in previous studies of subcellular localization was the degree of lysosome purification. In their early study of phosphatidylinositol ki- nase from rat liver. Michell et a1. (13) reported Pl kinase activity in a lysosome fraction that was enriched 4.28-fold over the homogenate based on B-glucuronidase relative specific activity. The relative specific activity of the Pl kinase was 0.59 in this fraction. These authors concluded that the enzyme distribution does not parallel that of nuclei. mitochondria. lysosomes. or endoplasmic reticulum. In an extension of this work. Harwood and Hawthorne (14) examined the PI kinase in rat liver. brain. kidney. heart. skeletal muscle. testis. and in human erythrocytes. In all cases they found the activity predominantly in fractions shown to be enriched in plasma membranes. However. they also detected a detergent-stimu- lated P1 kinase aetivity associated with crude lysosome and microsome fractions from rat liver and kidney. We report in this study the formation of phosphatidy lino- sitol 4-phosphste by the action of a lysosomal membrane- associated -J- ' ‘- ' kinase from highly purified rat liver lysosomes. Lil:e the Pl kinase activity reported tor microsomes of liver and Lidney (14 29) and microsomes and chromaffin granules from bovine adrenals (30). the lysosomal enzyme exhibited the highest specific aetin‘ty in the presence of e nonionic detergent and exogenous Pl (Table I). However. rapid degradation of the product. DPI. also occurred in the presence of Triton X-100. Preliminary experiments suggest 34 Lysosomal PI Kinase that degradation occurs through the action of both phospho- lipase C and phosphomonoesterase activities. There is no evidence to suggest that these enzymes are from the lysosome matrix. since degradation occurs at neutral pH. In addition. the acid phospholipase C in lysosomes has been reported to be unable to decade lysosomal membrane phosphatidylino- sitol (31). Polyphoephoinosiride phosphatases have been de- tected in both soluble and membrane fractions of rat kidney (32). Polyphosphoinositide-specific phosphodiesterase has also been reported to be both soluble and membrane bound (3. 34). Lysosomal PI kinase was inhibited by Ca" as has been reported for Other PI kinases (14. 30) and was also strongly inhibited by Le”. which is known to compete for Ca”-binding sites (35) (Table II). Unlike the enzyme activity reported in liver homogenates by Michell er al. (13). lysosomal PI kinase was not inhibited by the addition of other phospholipids. Phosphatidylinositol kinase was also found in this study to be associated with microsomes and plasma membrane (Table I). However. the high specific activity of the Triton-filled lysosome preparation and the occunence of PI kinase in normal liver lysosomes (Fig. 6) would argue that the phos- phatidylinositol kinase in our purified fractions is of lysosomal origin and not contamination from these other organelles. Studies with rat hepatocytes cultured in the presence of ”P. have shown that both TPI and DPI of lysosome fractions become labeled.’ Since only a small amount of labeled TPI was synthesized in isolated lysosomes. we speculate that a cytoplasmic diphosphoinoeitide kinase may be required to complete the sequential phosphorylation reactions observed in intact liver cells. A cytoplasmic location for this enzyme has been found in rat parotid gland (36) and brain (37). In rat kidney. DPI kinase was enriched in the Golgi complex but was also found in the plssrns membrane and supernatant fractions (29. 38). Stimulation of phosphoinositide metabolism has been ob- served in many different target tissues in response to agents which modify Ca" flux in the cell (10). Calcium appears to be involved in the regulation of phospholipase C activities that specifically degrade the ...:er r ' --"--- giving rise to diacylglycerol and inositol 1.4-bisphoephate and inositol 1. 4. 5- trisphosphate from DPI and TPI. respectively (34. 40-42). Hormones that mobilize Ca” in hepatocytes such as vaso- pressin. angiotensin. and epinephrine acting at a. receptors provide the rapid degradation of liver cell polyphosphoinosi- tides (43) It will. therefore. be of interest with respect to homonal control of autophagy and membrane fusion to de- termine whether the pol“ r m- in the lysosomal membrane are subject to the same regulation ‘e. a; ‘ We wish to acknowledge the expert amitance of Carol Fenn in the preparation of this manuscript. REFERENCES l 1. DeDuve. C. (1969) in Lysosomes in Biology and Pathology (Dingle. J. J.. and Fell. H. B.. eds) Vol 1. pp. 3-40. North- Holland Publishing Co.. Amsterdam Ashford. T. P.. and Porter. K. R. (1962) J. 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( 1967) Biochim. Biophys. Acta 144. 649-658 14. Harwood. J. L. and Hawthorne. J. N. (1969) Biochim Biophys. Acta 171. 75—88 15. Glynn. I. M.. and Chappell. J. B. (1964) Biochem. J. 90. 147-149 16. ReimannEM" Brostrom.C. 0.. Corbin.J. D.. King. C. A.. and KrebaE G. (1971)Biochern. Biophys. Res Commun.42.187- 17. Leighton. F. Poole. B. Beaufay. H.. Baudhuin. P.. Coffey. J W.. Fowler. S. andDeDuve. C (1968)J Cell Biol. 37. 482-512 18. Sellinger. O. 2.. Beaufay. H.. Jacques. P.. Doyen. A.. and DeDuve. C. (1960) Biochem. J. 74. 450-456 19. Fleischer. S.. and Fletcher. 3. (I967) Methods Enzyrnol. 10. 427- 428 20. Aronson. N 31. $402 21. Wattieua. R., Wattiaua-DeConinck. S.. Ronveaus-Dupal. M. P.. and DuBois P. ( 1978) J. Cell Biol. 78. 349—368 22. DeDuve. C., Pressman. B. C., Gianetto. R. Wattiaux. B... and Appelmane. F. (1955) Biochem. J. 80. 604-617 . Schecht. J. (1981) Methods Enzymol. 72. 626-631 Harmer. 6.. Eichberg. J.. and Gouda-Sane. F. (1971) Biochim. Biophys. Add as. 57-95 r. B. 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Jr., eds) pp. 383-397. Academic Press. New York . Kai. M., Salway, J. G.. and Hawthorne. J. N. (1968) Biochem. J. 108. 791-801 Tou. J.-S.. Hurst. M. W.. Huggins. C. 6.. and Foor. W. E. (1970) Arch. Biochem. Biophys. 140. 492-502 Deleted in proof. . Abdel-Latif. A. A.. Ahhtar. R A.. and Smith. J. P. (1978) in Cyclitols and Phoephoinositides (Wells. W. W. and Eisenberg. P.. Jr., eds) pp. 121-143. Academic Press. New York 1. Buckley. J. T.. and Hawthorne. J. N. (1972) J. Biol. Chem. 247. 7218-7223 42. Allen. D.. and Michell. R H ( 1978) Biochim. Biophys. Acta 808. 277-286 43. Kirk. C. J.. Michell. R. H.. and Heme. D. A. (1981) Biochem J. 194. 155-165 N., Jr., and Touster. O. (1974) Methods Enzymol. 313313 8.8.833 (1976) Can 88888823 8 A CHAPTER III CHARACTERIZATION OF AN ACYLPHOSPHATE INTERMEDIATE OF A LYSOSOMAL MEMBRANE ATPase 35 36 ABSTRACT Lysosomes prepared from liver of Triton-injected rats incorporate 32F from either [3-32PJATP or [3'-32P]GTP into a membrane protein of l80,000 daltons. The phosphate is present in an acyl linkage as deter- mined by sensitivity to alkaline pH and hydroxylamine treatment. Acyl- phosphate formation occurred in the abSence of a divalent metal cation, but the rate and extent of phosphorylation were increased in the presence of MgClz. Ca2+ did not stimulate 32P—incorporation and did not substitute for Mg2+ in the presence of the divalent metal chelator. trans-cyclohexane- l,2,-diamine-N,N,N',N'-tetraacetic acid (CDTA). However, dephosphorylation was stimulated by either Ca2+ or M92+. The rate of dephosphorylation in the presence of CDTA and a 50-fold excess of unlabeled ATP was found to be 0.2l s']. This dephosphorylation rate was equal to ATPase turnover as measured by'[K-32P]ATP hydrolysis in the presence of the chelator. Acyl- phosphate formation and the lysosomal membrane ATPase were similarly inhibited by dicyclohexylcarbodiimide, NaN3, fluorescein isothiocyanate, and sulfhydryl reagents. Na3VO4 was found to inhibit ATPase activity in a pH dependent manner. The lysosomal membrane preparation catalyzed phosphate exchange reactions between a nucleoside triphosphate and nucleoside diphosphate or Pi' These results suggest that the lysosomal membrane ATPase catalyzes cleavage of nucleotides by means of a phos- phorylated intermediate. 37 INTRODUCTION The existence of an ATP-dependent H+ pump on the lysosomal membrane has been postulated to account for the stimulation of proteolysis (l) and uptake of basic dyes (2), methylamine (3), and amino acid methyl esters (4) by lysosomes in the presence of ATP. Direct evidence for the existence of a lysosomal H+ pump has been obtained by Ohkuma gt;al, (5), who measured acidification of lysosomes filled with fluorescein isothio- cyanate-dextran by the change in fluoresence in response to ATP. An ATPase on the lysosomal membrane has been characterized (6) and it is suggested that this activity is functionally related to proton trans- location (3). The lysosomal ATPase and acidification properties are similar to the electrogenic proton pump ATPase of adrenal medulla chromaffin granules (7). A proton pump activity has also been identified in membranes of yeast vacuoles (8), secretory vesicles (9), and endocytic vesicles (l0). The ATPases of the lysosome and chromaffin granule are clearly distinct from the H+ pump ATPases of mitochondria, chloroplasts, and bacterial membranes (ll) in their lack of sensitivity to oligomycin and in their response to other inhibitors. Another class of H+ pump ATPases has been identified in the plasma membrane of fungal cells (l2). These enzymes are sensitive to the H+ pump inhibitor, DCCD, as are the other ATPases mentioned above (3, 7, ll). The fungal ATPase reaction procedes by means of an acylphosphate inter- mediate on a protein of l00,000 daltons (l3-l5). In this respect, its mechanism of action is similar to that of the cation pump ATPases of plasma membrane (l6-l9), sarcoplasmic reticulum (20, 21), and gastric 2+ mucosa (22). The Na+/K+ and Ca pump ATPases form a phosphorylated reaction intermediate on a l00,000 dalton subunit during the course of 38 ATP hydrolysis. These cation pumps are dependent on the transported ion for activity and are not inhibited by DCCD. In this Chapter, the characteristics of an acylphosphorylated protein in the lysosomal membrane are examined. Its properties suggest that it may be the catalytic intermediate of a lysosomal membrane ATPase. 39 EXPERIMENTAL PROCEDURES Materials - (32P)Orthophosphate, carrier free, and [I4CJADP (58 mCi/mmol) were purchased from ICN. [x-32PJATP and [x-3ZPJGTP were prepared by the method of Glynn and Chappell (23), as modified by [35$]Adenosine 5'-[r-thio]triphosphate (65 Ci/mmol) Reimann et_al. (24). was obtained from New England Nuclear. Unlabeled adenosine 5'-[r-thio]- triphosphate (ATPES) was obtained from Boehringer Mannheim. Other nucleotides, ATPase inhibitors, and lithium dodecylsulfate were purchased from Sigma. Cellulose (Avicel) thin layer plates were from Analtech. PEI-Cellulose coated plastic sheets and glass distilled organic solvents were from MCB. Reagents for polyacrylamide gel electrophoresis were purchased from Bio-Rad. Lysosome Preparation - Triton WR-l339 filled lysosomes were prepared from rat liver as described previously (25). Protease inhibitors, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor, were included in the homogenization medium at a concentration of l, 5, 75, and l00 mg/liter, respectively. Protease inhibitors were also added to the final lysosome preparation buffered at pH 7.5 with l0 mM Tris-MOPS. Lysosomal membranes were prepared by l cycle of freeze-thawing in hypoosmotic sucrose and collected by centrifugation at 80,000 x g for 45 min. The resulting pellet was resuspended in 10 mM Tris-MOPS, pH 7.5, 0.25 M sucrose and stored at -70°C. Membranes were also prepared by dilution of frozen lysosomes with an equal volume of 0.2 M NaCl, 50 mM Tris-MOPS, pH 8.0, l mM EDTA followed by centrifugation and resuspension of the pellet as before. Lysosomal membranes prepared in either manner contained 25% of the protein present in the intact lysosome fraction and 85% of the ATPase activity. 4O Phosphorylation Assay - The standard assay mixture contained 50 mM Tris-Cl, pH 7.5, 0.1 mM [t-32PJATP (500 - zooo cpm/pmol) and lysosomal membrane (l0 - 25 ug of protein) in a final volume of 50 ul. Additions of divalent metal cations and chelators were made as indicated in the figures. The reaction was initiated by the addition of ATP and incubation at 30°C was carried out for the times indicated. The reaction was quenched by adding l ml of cold l0% trichloroacetic acid containing 3ZP-labeled l0 mM NaPPi and 2 mM ATP. For zero-time controls the nucleotide was added after the trichloroacetic acid. Bovine serum albumin (20 pl of 5 mg/ml) was added as a carrier. The precipitates were collected on whatman GF/C filters and washed with l0 ml of l0% trichloroacetic acid containing NaPPi and ATP. The filters were dried and counted in 5 ml of scintillation fluid. Polyacrylamide Gel Electrophoresis - Low pH gels as described by Lichtner and Wolf (26) were prepared containing 7.5% acrylamide and 0.1% (w/v) LDS. The gel solution was buffered with 80 mM citric acid, l0 mM phosphoric acid adjusted to pH 3.5 with Tris base. The gels were polymerized with FeSO4 and H202 as detailed by Jones gt_al, (27). The electrode buffer contained 100 mM citric acid, l2.5 mM phosphoric acid, and Tris, pH 3.5, with 0.l% LDS. For analysis on polyacrylamide gels, membrane samples were phosphorylated as described above and precipitated by adding 0.5 ml of cold 10% trichloroacetic acid, 2 mM ATP. The protein was collected by centrifugation and washed once by resuspension in cold distilled water and centrifugation. The pellet was solubilized in 25 ul of lo mM Tris base containing 2% LDS. An equal volume of sample buffer containing electrode buffer, 20% (v/v) glycerol, 8% 2-ME, 4% (w/v) LDS, and methyl green as a tracking dye was then added. ~The gels were run at 40 mA for 4 h at 4°C. The gels were dried and exposed to Kodak XAR-S 4T x-ray film for autoradiography. Proteins used as molecular weight markers were run in a separate lane on the same gel and stained with Coomassie Blue. There was a linear relationship between log molecular weight and relative mobility on these gels in the range of 20 to l70 kilodaltons. ATPase Assays - l. Charcoal adsorption - The reaction mixture contained 50 mM Tris—Cl, pH 7.5, 3 mM [r-32PJATP (l-5 cpm/pmol), 5 mM MgCl2 or CaClZ, and sample (2-20 pg protein) in a final volume of 50 pl. 50 mM Tris-MOPS, pH 7.5 was used in experiments to study effects of salts on ATPase activity and did not affect the basal level of ATP hydrolysis. After incubation at 30°C the reaction was stopped by the addition of 0.5 ml of cold perchloric acid. After placing the tubes on ice, 300 pl of a suspension of acid washed charcoal was added. After l0 min on ice, 200 pl of a solution containing 5 mg of bovine serum albumin per ml and 50 mM NaPPi, pH 7, was added. The reaction tubes were vortexed and centrifuged at 2000 rpm in a GLC centrifuge for 5 min. Aliquots (0.5 ml) of the supernatants were removed andcxmnted directly by Cerenkov radiation with an efficiency of 35%. A blank incubation was included to correct 32 P. in the ATP preparation and for any for the slight amount of 1 degradation which occurred during the acid treatment. This value was generally l-3% of the total cpm added to the reaction. 2. Thin layer chromatographic analysis of reaction products - Assays were performed with labeled nucleoside triphosphate as described above but were terminated by the addition of $05 to a final concentration of l% (w/v). Aliquots (5 pl) of each reaction mixture were spotted on a cellulose thin layer plate which was then developed in n-butanolzacetic acidzH Ozpyridine, l5:3:l2:l0. The positions of the nucleotide and Pi 2 were located by autoradiography. The spots were scraped into 42 scintillation vials for counting. 3. Continuous spectrophotometric assay - ATPase activity was determined by measuring ADP formation in the presence of phosphoenol- pyruvate, NADH, lactate dehydrogenase and pyruvate kinase as described by Barnett (28) in a final volume of 0.5 ml. The disappearance of NADH was monitored by change in absorbance at 340 nm. NTP-NDP Phosphate Exchange - The phosphate exchange between ATP and ADP was measured as described by Makinose (20). The incubation mixture contained 50 mM Tris-MOPS, pH 7.5, 7 mM MgCl or 0.5 mM CDTA, 5 mM ATP, 2 2 mM [14C]ADP, and membrane sample (2-5 pg of protein) in a final volume of 50 pl. The reaction at 22°C was initiated by the addition of the nucleotides. The reaction was terminated by the addition of 0.5 ml of cold l0% trichloroacetic acid and 50 pl of 5 mg/ml bovine serum albumin. After centrifugation to remove precipitated protein, the supernatant was extracted twice with diethylether to remove the trichloroacetic acid. Aliquots of the supernatant and carrier nucleotides were spotted on PEI- cellulose sheets. After separation in 0.85 M KH2P04, pH 3.4, nucleoside triphosphates were visualized by ultraviolet light and cut out for scintillation counting. Control incubations without unlabeled ATP were included to correct for any adenylate kinase activity in the preparations. Exchange reactions utilizing GTP-ADP and ATP-GDP were performed as described above except that [f—32PJATP and [r-32PJGTP were used at a final concentration of 2 mM with unlabeled GDP or ADP at 2 mM. ATP-P, Exchange - The exchange between the terminal phosphate of ATP and Pi was measured according to Ronzani et al. (29). The reaction mixture contained 50 NM Tris-MOPS, pH 7.5, 5 mM ATP, 2 mM ADP, 7 nM MgCl2 or 0.5 mM CDTA, 5 mM [32P]potassium phosphate (200 cpm/pmol) and membrane sample (2-l0 pg protein) in a final volume of 50 pl. Incubation was 43 carried out after the addition of 32Pi for various times at 22°C. The reaction was terminated with 0.5 ml of l0% perchloric acid containing l mM phosphoric acid. After the addition of 0.5 ml of 2.5% ammonium molybdate, the phosphomolybdate complex was extracted 3 times with 2 ml of water—saturated isobutanol. Radioactivity in the remaining water phase was determined by Cerenkov radiation with a counting efficiency of 35%. To confirm that the radioactivity had been transferred to ATP, aliquots of the water phase were spotted on PEI-cellulose sheets and developed as described for the NTP-NDP exchange reactions. Protein Determination - Protein was determined by the method of Lowry (30) with bovine serum albumin as the standard. 44 RESULTS Substrate Specificity of the Acylphosphorylation Reaction - The time course of acylphosphate formation in lysosomal membranes is shown in Fig. l. The activity is similar using either [r-32PJATP or [K-BZPJGTP as phosphate donor in the presence of l00 pM CaClz. As demonstrated in Chapter I, this rapidly formed product is an acylphosphate based on its sensitivity to hot acid and pH values above 5. Much greaun~labeling 35S was observed using [3SSJATP8S as a substrate. Incorporation of reached a maximum by 2 min of incubation at 30°C and remained constant for at least 5 min. The 35S-labeled product showed a pH dependence of 32P-labeled product (25), with resistance to hydrolysis similar to the degradation between pH 0.5 and 6 (data not shown). Polyacrylamide Gel Analysis of the Acylphosphate - In a previous study (25 and in Chapter I), a 32 P-labeled product exhibiting properties of an acylphosphorylated protein was found to migrate at a position corresponding to l4,000 daltons in a cationic detergent gel system. 32P-labeled product was found at a Under similar assay conditions, a molecular weight of 180,000 in this experiment using LDS polyacrylamide gel electrophoresis. This product has also been identified as an acylphosphate based on its rapid turnover and degradation by hydroxylamine (Fig. 2). Quantitative analysis of the labeling of this radioactive band is shown in Table 1. Addition of a l00 fold excess of unlabeled ATP to the reaction mixture led to a 64% decrease in radioactivity in 5 s. Incubation of the labeled lysosomal membranes for 15 min with 2% LDS at pH 5.5 caused a slight reduction in radioactivity. Addition of hydroxyl- amine led to a further decrease in labeling of 36%.. No effect of hydroxylamine was observed in the absence of detergent (data not shown). 45 Figure l. Nucleotide Specificity of Phosphorylation Reaction. The phosphorylation of freeze-thaw prepared lysosomal membranes was carried out as in ”Experimental Procedures" with the inclusion of l00 pM CaCl2 and the indicated labeled nucleotide in the assay medium. O-O, [r-32PJATP; 0—0, [r-32PJGTP; A-A , [35$]ATPIS. The results are the mean of 2 experiments. 46 .85 05%220885 mnn_oEc occzu mpmcamoza N N .Aniuv Pom: :5 o ccm ap< :5 m Lo .Aaicv Fume :5 m use mh< :5 m .Aoiov mh< :5 m ”mczuxre cowpomwc asp Op mote mew: mcowpwvum mcvzo_Pow wgp .zoscm mg» as vmumowccw we?“ msp u< .occzp mumcqmoga .cowum—xcozamocawo soc pcmsmcwzcmm cow 98pm: .< .a weaved 54 @2583 22: q mgzmwu C) N) CD 00. AOON 4on bw/peioiodiooui dze'ow d 55 30 s. Substrate Specificity of the Lysosomal Membrane ATPase - ATP hydrolysis was measured by the three methods described under "Experimental Procedures". Each assay method gave comparable results in the determina- tion of ATPase specific activity. GTPase activity was determined by the charcoal adsorption and thin layer chromatography methods which gave identical results. GTP was also a good substrate and either Ca2+ or Mg2+ was an effective metal cofactor (Table II). Hydrolysis of the ATP analog, ATPtS, was very slow, exhibiting only 1% of the activity observed with ATP. ATPES hydrolysis was measured by the thin layer separation of ATPXS and thiophosphate and subsequent scintillation counting of these compounds. ATPJS is unstable under acidic conditions, therefore the charcoal adsorption assay could not be used. ADP determination was not as sensitive an assay because of the ADP present in the commercial ATPXS preparations. ATPase activity was also measured under the phosphorylation conditions described in Fig. 3, with 100 pM [X-BZPJATP. When added in concentrations in excess over CDTA, both Ca2+ and Mg2+ were effective in stimulating ATP hydrolysis. By 1 min of reaction at 30°C, 80% of the ATP was hydrolyzed. In the presence of 0.5 mM CDTA, ATP hydrolysis was reduced to 2 nmol/min/mg protein at 30°C. Addition of CDTA up to 5 mM did not reduce ATPase activity further (data not shown). Effect of Inhibitors on Acylphosphate Formation and ATPase Activity - To examine the relationship between ATPase activity and acylphosphate form- ation, several ATPase inhibitors were examined for their effect on these reactions (Table III). NaN3 was found to inhibit both activities, but only when the reaction took place in the presence of M92+. NaN3 did not inhibit either the ATPase or acylphosphorylation at up to 20 mM concentra- 2+ tion in the presence of Ca Both activities were inhibited by DCCD, 56 Table II. Substrate Specificity of Lysosomal Membrane ATPase.a Activity Nucleotide Mg2+ Ca2+ Mn2+ % ATP 100 105 54 GTP 90 89 50 ATPrS 0.26 0.63 1.1 aNTP hydrolysis was measured at 30°C in the presence of 3 mM nucleotide and 5 mM of the indicated metal ion as the chloride salt. The reactions were terminated at several time points up to 10 min to ensure linearity of the assay. ATP and GTP hydrolysis were measured by means of the charcoal adsorption assay. ATPES hydrolysis was determined by separating the reaction products on cellulose thin layer plates as described under "Experimental Procedures". The results are the means of at least 2 experiments ang+are expressed as a percent of ATP hydrolysis in the presence of Mg (1.2 pmol Pi/min/mg protein). 57 Table 111. Effect of Inhibitors on ATPase Activity and Acylphosphate Formation.a Additions ATPase Acylphosphate Activity Formation % None 100 100 NaN l3mM (Ca§:)b 102 - 20 mM (Ca ) 89 89 1 mM 76 95 5 mM 65 57 Quercitin, 120 pM 88 62 Diamide, 100 pM 89 93 DCCD 100 pH 80 - 200 pM 50 64 Ouabain, 1 mM 97 98 NaF, 1 mM 97 88 Na3V04, 1 mM 88 72 ADPC 63 62 Trimethyl tin, 100 pM 81 71 FITC, 100 pM 63 46 aLysosomal membrane samples were preincubated with the agents listed above for 30 min at 30°C. The reactions were initiated by the addition of [X-32PJATP and assays were carried out as described under "Experimental Procedures". ATPase activity was measured by charcoal adsorption assay after 5 min of incubation at 30°C in the presence of 5 mM MgC12 except where noted. Acylphosphate formation was measured after 15 s at 30°C by filter assay. The results are the means of at least 4 experiments and are expressed as the percent of the control activity without additions. Control ATPase activity is 950 nmol Pi/min/mg protein and acylphosphate formation is 360 pmol 2P incorporated/mg protein. b 2+ These assays were carried out in the presence of 5.mM Ca cl mM ADP was used for the ATPase assay, 0.1 mM for acylphosphorylation. 58 ADP, trimethyl tin, and FITC. Acylphosphate formation was more sensitive to quercitin than was the ATPase. The addition of 1 mM Na3V04 resulted in only slight inhibition. Diamide, ouabain, and NaF had no effect. The control activites were not affected by preincubation in the absence of inhibitor, nor by ethanol used as a solvent for some of the agents tested. The effect of vanadate on ATPase activity was examined further (Fig. 5). The degree of inhibition was found to depend on the pH of the assay. At pH 5, 1 mM Na3V04 inhibited ATP hydrolysis by 63% compared with 20% at pH 7.5. A similar inhibition was observed when ATPase activity was measured with the lower ATP concentration used for the phosphorylation assay. The greater sensitivity to vanadate at pH 5-6.5 cannot be due to inhibition of acid phosphatase or acid pyrophosphatase. The activity of these enzymes with ATP as substrate was measured at pH 5 under the conditions optimal for acid phosphatase (31), and was less than 100 nmol Pi produced/min/mg protein. Both the ATPase and acylphosphorylation activities were affected by sulfhydryl modifying reagents as shown in Table IV. NEM and pCMB were strong inhibitors, while addition of other agents had mixed results. In particular, the reduced sulfhydryl reagents cysteine, DTT, and 2-ME had little or no effect on ATPase activity byt inhibited acylphosphate formation. Various monovalent ions were tested for their effect on ATPase and acylphosphate formation in the presence of MgClZ. KCl, NaCl, and NH4C1 at 100 mM did not affect either activity. The addition of 1 mM ethylene glycol bis(fl-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) also had no effect on the Mg2+ stimulated activity (data not shown). Phosphoryltransfer Reactions - The lysosomal membrane preparation was examined for the presence of phosphaueexchange activities (Table V). 59 Figure 5. Effect of pH on Vanadate Inhibition of ATPase. ATPase activity was determined by the charcoal adsorption assay in the presence of Na acetate (pH 5), 2-morpholino)ethane- sulfonic acid (pH 6, 6.5), or Tris-Cl (pH 7-8) at a final concen- tration of 50 mM. The assay was carried out as described under "Experimental Procedures” in the absence (O-O), or presence (O-O), of 1 mM Na3V04. 60 _ \o \o 18 A \o ./ O/ 17 O O H / / D. O O 16 o o 15 _ _ _ _ O O O O o o o o o s 8 6 4 2 m 520.5 955.55 .06: W 61 Table IV. Effect of Sulfhydryl Reagents on ATPase Activity and Acylphosphate Formation.a Addition ATPase Activity Acylphosphate Formation % None 100 100 NEM 62 33 pCMB 76 63 GSSG 57 61 GSH 60 59 Cystine 88 113 Cysteine 93 50 DTT 102 69 2-ME 90 41 aATPase assays and acylphosphate formation were carried out as described for Table III. The additions were made to give a final concentration of 1 mM, except to pCMB and 2-ME which were 0.1 mM and 5 mM, respectively. After preincubation for 30 min at 30°C, [r-3ZPJATP was added to start the reaction. The results are the means of 2 experiments and are expressed as percent of control activity. 62 Phosphoenzyme formation and exchange reactions were performed as described under "Experimental Procedures". ATPase activity was analyzed by the charcoal adsorption assay. Phosphate transfer was demonstrated between NTP and NDP or Pi' Exchange activity between NTP and NDP was less with GTP as the phosphate donor than with ATP. NTP-NDP exchange reactions occurred to almost the same extent in the absence of metal (presence of 0.5 mM CDTA). Phosphate exchange between ATP and 32 Pi was much lower than NTP-NDP exchange, however the activity was 4 fold higher in the absence of metal ion than in the presence of 7 mM MgC12. 63 Table V. Phosphoryl Transfer Reactions of the Lysosomal Membrane ATPase.a Transfer Activity Reaction Mg2+ CDTA nmol/min/mg protein Phosphoprotein formationb (4) 0.26 0.16 ATPase (4) 1010 7.4 NTP-NDP exchange GTP-ADP (4) 29.0 20.0 ATP-GDP (2) 176 76 ATP-ADP (2) 150 ll6 ATP—P1 exchange (4) 0.42 1.82 aThe reactions indicated above were carried out in the presence of MgCl2 or 0.5 mM CDTA as described under “Experimental Procedures”. The values in parentheses give the number of determinations. b 32P incorporation in nmol/mg protein after 30 s of incubation. 64 DISCUSSION The acylphosphate product identified in Chapter I has been further characterized with regard to hydroxylamine sensitivity, substrate specificity, metal ion requirement, and effect of inhibitors. Acyl- phosphate formation appears to be associated with a lysosomal membrane protein since its specific activity is increased in membrane preparations over that observed with intact lysosomes. The acylphosphate is not produced by the action of Na+/Kf or Ca2+/Mg2+ ATPase since the Triton— filled lysosome preparation contains little contamination by either plasma membrane or microsomes. In addition, acylphosphate formation 2+ or Na+ and KI in a manner consistent with the was not affected by Ca activity of these other acylphosphate forming ATPases. Hydroxylamine-dependent loss of label has been accepted as an indication of the presence of an acylphosphate bond (31). It was found that hydroxylamine treatment had no effect unless 2% LDS was included in the incubation mixture. This resistance of an acylphosphate to hydroxylamine has also been found for a phosphoprotein intermediate from Golgi vesicles (33), and suggests that the acylphosphate is not readily accessible to the medium. The molecular weight derived from LDS gel electrophoresis can be regarded as only approximate. The major difficulty in the gel analysis appears to be the inadequate solubilization of the membrane material under the conditions required to minimize proteolysis and chemical breakdown of the acylphosphate. In most experiments, the radioactivity remained at the top of the gel. Nonionic detergents, urea, and organic solvents have been added to the sample buffer to solubilize the proteins with limited success. In some experiments, however, analysis of the 65 32P-labeled membranes on LDS gels at pH 3.5 showed a high molecular weight band (Fig. 2). The previously observed 14,000 dalton band (Chapter I) was not detected in this experiment. The kinetics of acylphosphate hydrolysis in this earlier study suggested that only one species was present in the lysosomal membrane. The time course of formation and the pH profile of hydrolysis of the labeled product in the present study were found to be similar to that described in Chapter 1, suggesting that the phosphorylated species detected by filter assays in each case were the same. It is possible that the 14,000 and 180,000 dalton species are related and that proteases known to be active in membrane preparations even in the presence of protease inhibitors (3, 34) are responsible for the formation of the lower molecular weight product demonstrated earlier. More recent preparations have utilized 2 additional protease inhibitors, leupeptin and pepstatin, which were not present for the studies shown in Chapter I. The cationic detergent, tetradecyltri- methyl ammonium bromide, may not completely inactivate proteases in the lysosomal membrane during sample preparation and electrophoresis. Lithium dodecylsulfate may reduce proteolytic activity so that the higher molecular weight species can be detected. Attempts to induce the formation of a labeled 14,000 dalton product by the omission of protease inhibitors have been unsuccessful, however. Acylphosphates of approximately 100 kilodaltons in other membrane systems have been identified as the catalytic intermediate of a cation pump ATPase (16-21). Their identification was facilitated by the requirement for the transported ion for activity of both the ATPase and acylphosphorylation reaction. However, acylphosphate intermediates have also been identified for the proton pump ATPases of yeast and neurospora plasma membranes (12-15). Several studies of the lysosomal proton pump 66 have been carried out (4, 5, 34, 35), and suggest that a lysosomal membrane ATPase is responsible for this activity (3). The properties of the acylphosphorylation reaction were examined to see if there was a correlation between this activity and the ATPase reaction. GTP and ATP are good substrates for both acylphosphate formation (Fig. l) and NTPase (Table II). Both Ca2+ and Mg2+ will promote lysosomal ATPase or GTPase activity, as has been observed previously (6). GTP has also been found to promote lysosome acidification, although to a lesser extent than ATP (3-5). Ca2+, however, will not substitute for M92+ in this activity (5). The ATP analog, ATPXS, was examined as a possible substrate for acylphosphorylation since it is resistant to phosphatase action (36-38). Incorporation of 2 nmol 35S/mg protein into trichloroacetic acid precipitable material was found (Fig. l). The pH stability of the thiophosphate bond was consistent with this product containing an acyl linkage. To examine whether this analog was hydrolyzed by the lysosomal membrane ATPase, assays were performed as shown in Table II. The low hydrolysis rate and the high level of thiophosphorylation suggest that the turnover of the acyl-intermediate is extremely slow. Therefore, the extent of 35S-incorporation may represent the total number of acyl- phosphorylation sites on the lysosomal membrane. The experiments described in Figures 3 and 4 were designed to examine the metal ion requirement for acylphosphate formation and turn- over. As shown in Fig. 3, acylphosphate formation was not completely dependent on the presence of a divalent cation. The addition of Mg2+ increased both the rate and extent of phosphorylation, while Ca2+ addition led to a slower 32F incorporation. Similar results have been observed for phosphoenzyme formation in the red blood cell Ca2+ 67 ATPase (39). Acylphosphate formation occurred in the absence of metal with MgZ+-depleted red cell membranes, although the level was only 50% of that with added Mgz+. The dephosphorylation rate was also much slower in the absence of metal. In studies with the Na+/K+ ATPase, Ca2+ was used to replace Mg2+ in order to decrease the rate of phosphoenzyme formation (41). The steady state level of the phosphorylated inter- mediate was also lower with Ca2+, although Ca2+ will replace Mg2+ for ATP hydrolysis by this enzyme (42). These results in other systems are similar to what was found here for the lysosomal membrane activity. Although free ATP will act as a substrate for the Ca2+ pump ATPase at low concentrations (40), it is likely that the MgZ+-ATP complex is the physiological substrate. Other studies of ATPases which form an acyl- phosphate intermediate have demonstrated a requirement for a divalent 2+ bound to cation, usually MgZ+, which can be met by the low levels of Mg membrane and in the assay medium. Acylphosphate formation occurred in the presence of 0.5 mM CDTA in lysosomal membranes which had been prepared in the presence of 1 mM EDTA. Although tightly bound Mg2+ may still be present in the membranes under these conditions, this possibility seems unlikely. It is possible, however, that low amounts of Mg2+ bound to membrane or in the assay medium in the absence of CDTA account for the 2* (Fig. 1) compared with Ca2+ in the presence of CDTA (Fig. 3). The similar increased rate and extent of acylphosphate formation with Ca phosphorylation rates observed with either Ca2+ or Mg2+ in Chapter I can also be explained by the presence of endogenous metal ion.' Phosphoryla- tion experiments performed in the absence of chelator and added divalent 32F incorporation can be obtained metal cations indicate that maximal under these conditions, as well. For the red cell Ca2+ ATPase, Mg2+ at 0.5 mM increases both the 68 rate and extent of phosphoenzyme formation, with a concommitant increase 2+ has been proposed to in the rate of dephosphorylation (19, 43). Mg increase the rate of hydrolysis of the acylphosphate and therefore increase the ATPase activity. Since maximal rates of lysosomal membrane ATP hydrolysis require a metal cation, the effect of Ca2+ and Mg2+ on the dephosphorylation reaction was examined. Acylphosphate formation was allowed to procede until maximal incorporation of 32F occurred in the presence of 100 pM Ca2+ and, presumably, pM amounts of Mg2+ as in Fig.1. Addition of 1 mM CDTA at this point stabilized the phosphoenzyme, whereas the control acylphosphate was rapidly dephosphorylated (data not shown). This is consistent with a mechanism requiring metal for rapid turnover to occur. The dephosphorylation reaction was also examined in membranes labeled to steady state in the presence of CDTA. Under these conditions, ATP hydrolysis is very slow, only 2.2 nmol Pi produced/min/mg protein at 30°C. The addition of 5 mM unlabeled ATP or 5 mM ATP and 6 mM Ca2+ led to a rapid loss of label from the protein. This rate of dephosphoryla- tion was similar to the rate of ATP hydrolysis (26 pmol/s vs 37 pmol/s) assuming immediate dilution of the ATP label and constant turnover of the acylphosphate. Therefore, the addition of ATP does not stimulate the dephosphorylation reaction. Instead, the loss of label is due to turnover of the enzyme in the presence of unlabeled ATP. Addition of unlabeled ATP with Mg2+ had a different effect, however. Dephosphorylation occurred more slowly than with ATP alone or with Ca2+. This is also observed in Fig. 48, where M92+ is added without unlabeled ATP. After a slow period 32P incorporation was observed. Since 2+ of dephosphorylation, increased unhydrolyzed [r-32PJATP is present in the assay medium, Mg addition stimulates acylphosphate formation beyond the level observed in the presence of CDTA. Even with an excess of unlabeled ATP as in Fig. 4A, 69 addition of Mg2+ may stimulate acylphosphate formation by [x-32PJATP bound by the enzyme which may not be readily exchangeable by added ATP. The actual metal requirement for dephosphorylation is difficult to assess with these limited number of experiments. However, both Ca2+ and Mg2+ appear to stimulate the dephosphorylation reaction (Fig. 4B). This suggests that although metal is not absolutely required for acyl- phosphate formation, rapid ATP hydrolysis will only occur in the presence of a divalent metal cation. The results of experiments examining the 2+ and Mg2+ on acylphosphate formation and hydrolysis are effects of Ca summarized in Table VI. The data are consistent with the dephosphoryla- tion of the acylphosphate intermediate being the rate limiting step 2+ in the H+ for ATP hydrolysis. The inability of Ca2+ to replace Mg pump activity of the lysosomal ATPase may be related to the different effects these ions have on acylphosphate formation. ATP hydrolysis in the presence of Ca2+ may be ”uncoupled” from the lysosome acidification observed in the presence of M92+ and ATP. In Tables III and IV, the effects of various ATPase inhibitors were tested for their effects on the lysosomal membrane ATPase activity and acylphosphate formation. DCCD has been reported to inhibit ATP— dependent acidification of lysosomes (3, 5, 34, 35) as well as other proton pumps (7-12), but the inhibition of ATPase activity is less than the effect on acidification (3). DCCD and NaN3 have been found to inhibit the H+ pump activity of Triton WR-1339 prepared lysosomes but not normal lysosomes (5). Inhibition by NaN3 was found to be dependent on the metal ion cofactor. Both the ATPase and acylphosphate formation were 2+ inhibited by NaN3 in the presence of MgZ+, but not Ca No other difference between MgZ+ and Ca2+ stimulated ATPase activity was found. The different effects of Ca2+ and M92+ on acylphosphate formation have 70 o» wasnwcucou xwe cowpmcmamca AcmanmE asp cw ucmmmca . 9:5.wa cowuwnzucw +N .mupammc as“ a: om pcmswcmaxm Avg» com com: go: no: <~au a .vmumowvcw mm wu mumcwswpm op ucmmmca mm; mpnmw 71 already been discussed. Quercitin is a strong inhibitor of lysosome acidification (5). Only a slight effect of this reagent on ATPase activity was found. Trialkyl tin has been shown to inhibit the H+/K+ exchange of yeast membranes (44) and the proton pump of mitochondria (45). Again, only a slight inhibition was demonstrated in the presence of this compound. FITC has been used to modify the active site of sarcoplasmic reticulum Ca2+ ATPase and the Na+/K+ ATPase (46). It was shown that FITC dose- dependently inhibited both ATPase activity and acylphosphate formation, indicating a functional relationship between the two reactions. FITC was shown here to inhibit both activities, as well. Inhibition by NEM and pCMB suggest a role for sulfhydryls at the active site, as has been found for other ATPases (47). NEM has also been shown to inhibit the lysosomal proton pump activity (5). 0n the basis of these results, the lysosomal membrane ATPase cannot be definitively classed with either the H+ translocating ATPases of mitochondria and yeast membranes, or the cation pump ATPases referred to above. However, the similar degree to which both the ATPase activity and acylphosphate formation are inhibited by these agents suggests that there may be a functional relationship between them. Inhibition by sodium orthovanadate has been taken as evidence that an ATPase reaction procedes through an acylphosphate intermediate (48). However, the concentration of vanadate required for ATPase inhibition ranges from several micromolar to 1 mM or more (12, 49). In other systems, inhibition by vanadate is influenced by nucleotide concentration, Mg2+ concentration, and is strongly dependent on pH (50, 51). Lack of inhibition by vanadate under only one assay condition is not a sufficient criterion for ruling out the possibility of a covalent intermediate in an 72 ATPase reaction. In at least one case (15), acylphosphate formation was unaffected by 500 pM vanadate even though ATPase activity was inhibited by 70%. Under different assay conditions the acylphosphate formation was found to be inhibited as well (49). It was found here that acylphosphate formation in the lysosomal membrane was inhibited by 28% in the presence of 1 mM Na3V04 at pH 7.5. ATPase activity was examined as a function of pH in the presence of 1 mM vanadate (Fig. 5). As has been previously described for other H+ pump ATPases (50, 51), the greatest inhibition was observed at slightly acidic pH. Lysosomal acidification was found not to be inhibited by vanadate (4, 5, 35). However, these experiments were conducted at pH 7 where little inhibitory effect on ATPase activity is observed. The ATPases involved in the transport of Na+/K+ and Ca2+ across membranes have been shown to procede by a mechanism involving one or more phosphoenzyme intermediates. Under certain conditions, reversal of the reaction sequence can lead to phosphate exchange between nucleo- tides and inorganic phosphate and from nucleoside triphosphates to nucleoside diphosphates (41, 52, 53). The lysosomal membrane was examined for the presence of these phosphoryltransfer activities. Exchange of the gamma position phosphate from NTP to NDP was found to occur independently of the presence of metal ion. ATP-ADP exchange occurred as rapidly as the ATP-GDP reaction. GTP, however, was only 20% as effective as ATP as a phosphate donor. This may be related to the fact that ATP is three times more effective than GTP in supporting lysosomal acidification, although the hydrolysis rates found here were similar. This substrate specificity has been observed for the exchange reactions of the sarcoplasmic reticulum Ca2+ ATPase, even though the hydrolysis rates of ATP and GTP for this enzyme are also similar (29). 73 Results with the Na+/KI ATPase demonstrate that GTP uncouples hydrolysis from transport, even though GTP is hydrolyzed as well as ATP under normal assay conditions (54). The NTP-NDP exchange reactions did not require the presence of a divalent cation. This was somewhat expected since the formation of the phosphoenzyme occurred in the presence of CDTA, and the exchange occurs as a result of the reversal of this reaction by the addition of NDP. Nucleoside diphosphate kinase is therefore not responsible for carrying out the exchange activity observed here, since M92+ would greatly stimulate this enzyme. The Pi-ATP exchange is due to the reversibility of the last step in the reaction sequence leading to hydrolysis of the acylphosphate and formation of P1. The rate of this exchange reaction was much lower than that observed for NTP-NDP exchange. The fact that this reaction does occur implies that a phosphoenzyme intermediate may be formed by incubation of the lysosomal membrane with 32 Pi- Preliminary results suggest that this does occur, however, the extent of labeling is very low. ATP-Pi exchange was 4-fold faster in the absence of metal than with 5 mM Mg2+ It is not known whether this reflects a change in affinity of the enzyme for P1 or is a consequence of Mg2+ stimulation of the forward reaction, thereby decreasing the ability of the enzyme to catalyze ATP-Pi exchange. The occurrence of these exchange reactions in the lysosomal membrane suggests that an acylphosphate intermediate is involved in the hydrolysis of nucleoside triphosphates. The ATPase activity of the lysosomal membrane is responsible for catalyzing the transport of H+ to acidify the interior of the organelle. This H+ pump may therefore be similar to the H+ translocating ATPase of fungal membranes in the formation of a covalent phosphoprotein intermediate in the enzyme reaction mechanism. ll. 12. 13. 14. 15. 16. 17. 18. 19. 20. 74 REFERENCES Mego, J.L., Farb, R.M., and Barnes, J. (1972) Biochem. J. 128: 763-769. Dell'Antone, P. (1979) Biochem. Biophys. Res. Commun. 86: 180-189. Schneider, D.L. (1981) J. Biol. Chem. 256: 3858-3864. Reeves, J.P., and Reames, T. (1981) J. Biol. Chem. 256: 6047-6053. Ohkuma, S., Moriyama, Y., and Takano, T. (1982) Proc. Natl. Acad. Sci. USA 79: 2758—2762. Schneider, D.L. (1977) J. Membrane Biol. 34: 247-261. Johnson, R.G., Beers, M F., and Scarpa, A. 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The results presented here do not provide any evidence for this mechanism. The possibility remains that cytosolic protein kinases may interact with membrane substrates, in vivo. The data in Chapter I indicate that lysosomal membrane proteins are not substrates for cyclic AMP—dependent kinase, however other soluble kinases or effectors could be involved. These studies did reveal the presence of two previously unreported enzyme activities in lysosomal membrane preparations. Phosphatidyl- inositol kinase had been thought to be located primarily on the plasma membrane. This activity is involved in the polyphosphoinositide cycle, which is stimulated in many different cell types in response to hormones which utilize calcium as a second messenger. The functions of the polyphosphoinositides in honnone action is unknown, although it has been suggested that polyphosphoinositide breakdown is coupled to calcium gating at the plasma membrane. It would be of interest to determine whether lysosomal polyphosphoinositide metabolism is affected by cx-adrenergic agonists, or by insulin and glucagon, which also have effects on calcium flux in the cell. The presence of phosphatidyl- inositol kinase in plasma membrane, endoplasmic reticulum, lysosomes, and chromaffin granules may result from the common origin of these 77 78 membranes from the GERL (Golgi-endOplasmic reticulum-lysosomes) complex of the cell. It is not known whether the Golgi apparatus or secretory vesicles also derived from this complex contain polyphosphoinositides. It is likely that the polyphosphoinositides can exert a large effect on local membrane structure, even though they may be present in low concentrations. The phosphates on the inositol head group may bind divalent metal ions. This greatly affects the hydrophobicity of the lipid, allowing it to move in the plane of the membrane. These lipids may also be clustered in certain regions of the lysosomal membrane, binding tightly to specific proteins such as the ATPase. The regula- tion of the enyzmes involved in polyphosphoinositide breakdown and synthesis as well as the function of these lipids in the cell remains to be determined. The second enzymatic activity in the lysosomal membrane catalyzes the incorporation of 32F from [X-3ZPJATP into an acyl linkage. The labeled material migrates with an approximate molecular weight of 180,000. Almost all examples of acylphOSphate-containing proteins are cation pump ATPases, the exceptions being acetate kinase from bacteria and ATP citrate lyase from rat liver. Although there is no known NaT/K+ or Ca2+ ATPase in the lysosomal membrane, there is an ATPase which has been identified as a proton pump. The multisubunit proton pump ATPases of bacteria, chloroplasts, and mitochondria do not procede through an acylphOSphate intermediate. The proton translocating ATPases of fungal cells, however, do have covalent intermediates, and have protein subunit compositions similar to those of the cation pumps. A third class of ATP-dependent proton pumps includes those of lysosomes, chromaffin granules, other secretory vesicles, yeast vacuoles, acrosomes, and endosomes. These enzymes have been thought 79 not to go through a covalent reaction intermediate, but this has not been rigorously tested and the ATPases have not been purified. Although there are multiple phosphatase activities in the lysosomes, the properties of the acylphosphate described in Chapter III are most consistent with those of the proton pump ATPase. More careful analysis of the ATPase and its regulation requires purification and reconstitu— tion of the protein into lipid vesicles in order to demonstrate proton translocation and to determine whether a phosphoenzyme form exists. ”'11111111111111111111111111111111”