A STUDY OF SUPRAMOLECULAR ORGANIZATION OF GLYCOGENOLYTIC ENZYMES IN VERTEBRATE MUSCLE TISSUE. V. K. Shmelev and T.P. Serebrenikova

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1 Vol. 43, No. 4, November ] 997 BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL Pages A STUDY OF SUPRAMOLECULAR ORGANIZATION OF GLYCOGENOLYTIC ENZYMES IN VERTEBRATE MUSCLE TISSUE V. K. Shmelev and T.P. Serebrenikova I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez prospect 44, St. Petersburg , Russia Received September 1, 1997 SUMMARY: Under conditions preventing the direct binding of phosphorylase kinase to glycogen, we detected the formation of the compound ternary complex of glycogen, phosphorylase and phosphorylase kinase. The complex formation occurs in two stages: (i) the formation of phosphorylase-glycogen complex controlled by ATP, (ii) the binding of phosphorylase kinase to the previously formed phosphorylaseglycogen complex exclusively in the presence of Ca z+ and Mg 2+. The process is responsible for the increase of phosphorylase kinase activity in the presence of glycogen. An opinion is offered that a specific order of enzyme binding to glycogen particle as support provides for a self-assembly of the studied metabolon and plays an essential role in the regulation of glycogenolysis. KEY WORDS: Supramolecular Organization, Glycogen, Phosphorylase, Phosphorylase Kinase INTRODUCTION Organization of enzymes on cellular organdies involving formation of ordered supramolecular structures is of great importance in the regulation of cellular metabolism (1-4). In the muscle tissue of vertebrates glycogenolytic enzymes are adsorbed on glycogen granules (5). The binding of glycogen phosphorylase b (EC ) to glycogen has been studied in detail (6,7). The presence of a specific glycogen storage site in the phosphorylase molecule has also been shown (8). The interaction of glycogen phosphorylase b kinase (EC ) with glycogen is studied less thoroughly. Steiner and Marshall have shown that under certain conditions phosphorylase kinase and glycogen form a complex compound and this process is accompanied by local conformational changes in the enzyme molecule (9). The stimulation of the process of phosphorylase b activation by phosphorylase kinase in the presence of glycogen might be due to this interaction (10). However, an alternative mechanism is possible: the affinity of phosphorylase kinase for phosphorylase-glycogen complex might increase as compared with the flee phosphorylase (11,12). The regular features of the structure organization on the glycogen particle as support with the involvement of several enzymes have not been investigated /97/ /(3 Copyright by Academic Press Australia, All rights of reproduction in any form reserved,

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL To understand the mechanism of the formation of the ordered supramolecular structure on the glycogen particle we investigated the complex formation of

2 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL To understand the mechanism of the formation of the ordered supramolecular structure on the glycogen particle we investigated the complex formation of rabbit phosphorylase kinase and phosphorylase b of some animal species with glycogen, and the influence of these processes on phosphorylase kinase activity. MATERIALS AND METHODS Phosphorylase b kinase was purified from rabbit skeletal muscles according to Cohen (13) using the ion exchange chromatography on DEAE-Toyopearl at the last step (14). Rabbit skeletal muscle phosphorylase b was prepared by the method of Fischer and Krebs (15). Skeletal muscle phosphorylase b of frog Rana temporaria and phosphorylase b of cartilaginous fish - thomback ray Raja clavata were prepared as described elsewhere (16). Phosphorylase kinase activity was determined by the method of Cohen (13), phosphorylase activity by the method of Cori as modified for ectothermic animals (17). Protein concentration was assayed by the method of Peterson (18). In the experiments on the binding ofphosphorylase kinase to phosphorylase-glycogen complex we performed as a preliminary the formation of phosphorylase-glycogen complex in the presence of ATP (the first stage of the process). The intensity of the complex formation of phosphorylase or phosphorylase kinase alone with glycogen was assayed from the increase in turbidity of the reaction mixture (9), using a spectrophotometer Specord M-40 (Carl Zeiss, Jena, Germany). The intensity of the phosphorylase kinase binding to the phosphorylase-glycogen complex (the second stage of the process) was estimated from an additional increase in turbidity of the incubation mixture upon addition of Ca 2+ and Mg z+ (9). The complex formation of the enzymes with glycogen and the dependence of phosphorylase kinase activity on the glycogen concentration were studied in the conditions of phosphorylase kinase assay (13). The phosphorylase kinase concentration in reaction mixture was 0,03 mg/ml in the activity assay whereas 0,2 mg/ml in the complex formation experiments for the better sensitivity of the method. Comparing the results obtained we estimated in relative units Y the intensity of enzyme-glycogen complex formation and the activity of phosphorylase kinase upon addition of glycogen. Y was the ratio of the complex formation intensity or the phosphorylase kinase activity to the corresponding value at saturating glycogen concentrations. To determine the kinetic constants, the linear regression of the data in the coordinates {[G]0/3(; [G]o } was performed using Sigma Plot (Jandel Scientific) program, where [G]0 was the glycogen concentration Dithiothreitol and Norit A were purchased from Serva, a-glycerophosphate and 13- glycerophosphate from Merck (Germany), 13-mercaptoethanol from Loba chemie (Austria), ATP, EDTA, glucose-l-phosphate and DEAE-cellulose from Reanal (Hungary), other reagents (of high purity) were from Russian suppliers. Pig liver glycogen was from Olaine plant (Latvia). RESULTS To study the supramolecular organization of glycogenolytic enzymes we investigated the processes of interaction of the components in the system consisting of glycogen, rabbit phosphorylase kinase and phospho~ylase b of rabbit, frog or fish (thomback ray). Building the model one should take into account two different interactions: (i) the direct phosphorylase kinase complex formation with glycogen and (ii) the binding of phosphorylase kinase to the previously formed phosphorylase b - glycogen complex. The phosphorylase kinase-glycogen complex 868

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL formation is the most intense in HEPES buffer (curve 1 in Fig. 1, a). The increase of ionic strength, which is more relevant to physiological conditions, makes the process difficult, while the addition of 0.2 M of NaCI or KC1 stops it completely. We did not practically observe the direct interaction of rabbit phosphorylase kinase with glycogen (curve 4 in Fig. 1, a) in the conditions of phosphorylase kinase assay (13). In this case, however, the enzyme could bind to the previously formed phosphorylase-glycogen complex, thus forming a ternary glycogen-phosphorylase-phosphorylase kinase complex (Fig. 1, b). The formation of the temary glycogen-phosphorylase-phosphorylase kinase complex occurs in two stages: (i) the formation of phosphorylase-glycogen complex controlled by ATP, (ii) the phosphorylase kinase binds to the previously formed phosphorylase-glycogen complex exclusively in the presence of Ca 2+ and Mg 2+ (19). For phosphorylase b from vertebrate species (rabbit, frog or fish) examined we obtained the similar character of phosphorylase kinase activation by glycogen and phosphorylase saturation with glycogen in the presence of ATP. The linear anamorphoses (Fig. 2) show coincidence of kinetic constants of both these processes in rabbit and thomback ray (p<0.01). The apparent binding constant with glycogen Kb of rabbit phosphorylase and the apparent activation constant K~ of phosphorylase kinase for glycogen are equal to 0.08 q (g/liter) and (g/liter), respectively. In the case of fish phosphorylase values of 0,10 AA 1,0 Y 0,05 ~ 0,5 0,00 0,0 r t, min [Glo, gh Fig. 1. Binding ofphosphorylase kinase to glycogen(a) and to previously formed phosphorylase-glycogen complex (b). (a) Complex formation ofphosphorylase kinase (0,2 mg/ml) with glycogen (0,2 g/liter) on the time of incubation in different conditions: 50 mm HEPES buffer (1), 50 mm tris-maleate (2), 50 mm tris- HCI (3); in the conditions of phosphorylase kinase assay in the absence of phosphorylase (4) and in the presence of 0,6 mg/ml phosphorylase (5). (b) The dependence of the saturation of phosphorylase kinase (0,2 mg/ml) with the previously formed phosphorylase-glycogen complex on the glycogen concentration [G]0 in the conditions of phosphorylase kinase assay. 0,6 mg/ml phosphorylase. Experimental data are presented in the coordinates {Y, [G]0} as discussed under Materials and Methods. 869

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL [G]/Y 1,0 1~/~ IGIdY 6,0 1 0,8 O 0,6 0,4 0,2 J I I I t -0,2 0,0 0,2 11,4 0,6 0,8 1,0 IGI., ~ 3, b I I ] I [ IGI~ g/l Fig. 2. The processes of the phosphorylase saturation with glycogen and the phosphorylase kinase activation by glycogen for phosphorylase from different species: rabbit (a) and thomback ray (b). 0,6 mg/ml phosphorylase, 0,03 mg/ml phosphorylase kinase. The dependence of the saturation of phosphorylase with glycogen on the: 1, in the absence of ATP (O); 2, in the presence 0,1 mm ATP ([~). The dependence of the phosphorylase kinase activity on the glycogen concentration (3, A). Experimental data are presented in the coordinates {[G]0/Y, [(3]0} as discussed under Materials and Methods. calculated constants Kb and K, are equal to (g//iter) and 0.65 _+ 0_08 (g/liter), respectively. It should be noted that the constants Kb without ATP are considerably different for the rabbit and fish phosphorylases and are equal to 0.20 _ and (g/liter), respectively. The same calculation for frog phosphorylase is impossible due to kinetic anomaly (Fig. 3). DISCUSSION For all the species examined the phosphorylase-glycogen complex formation may be assumed to be the crucial factor in the studied processes; the increase in phosphorylase kinase activity is proportional to the amount of the phosphorylase-glycogen complex that is phosphorylase kinase substrate. Phosphorylase conformation is known to change when it binds to glycogen. Phosphorylase kinase, apparently, has greater affinity for the modified phosphorylase, rather than for the flee enzyme. Addition of glycogen does not diminish K= value (20) when a synthetic peptide is used as a substrate and the mentioned molecule reorganization is thus ruled out. When the phosphorylase kinase binds to phosphorylase as a component of the phosphorylase-glycogen complex, the question remains whether the phosphorylase kinase binds directly to glycogen herein. It seems likely that the binding of phosphorylase kinase to phosphorylase as a component of phosphorylase-glycogen complex is, in turn, accompanied by a conformation change of 870

5 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 1,0 Y 1,0 Y 0, ,5 0, [G]o,g/I a b 0, IG]o, g/i ~ig. 3. Saturation of frog phosphorylase with glycogen and activation ofphosphorylase kinase by glycogen. ['he dependences of the frog phosphorylase complex formation with glycogen (a) and the phosphorylase "dnase activity (b) on the glycogen concentration [G]0: 1, in the absence of AT/) (O); 2, in the presence 0,1 nm ATP ([]). 0,6 mg/ml phosphorylase, 0,03 mg/ml phosphorylase kinase. Experimental data are )resented in the coordinates {Y, [G]0} as discussed under Materials and Methods. phosphorylase kinase molecule. This change makes the enzyme-glycogen binding sites accessible, that leads :o formation of the compound ternary glycogen-phosphorilase-phosphorilase kmase complex and as result Lo increase in the activity of phosphorylase kinase. The data of Chan and Graves (21) show that phosphorylase kinase molecule actually has a glycogen binding site, presumably located on ot-subunit, since the stimulating effect of glycogen on phosphorylase kinase activity is completely lost when this subunit is removed. It can, therefore, be assumed from our experimental data that there is a certain order of enzyme binding toglycogen particle as support, namely, the order of the self-assembly of this metabolon. The found experimental conditions, when the direct interaction of phosphorylase kinase with glycogen is impossible, can be used in building model of the assembling of glycogenolytic enzymes and glycogen and studying its conditions and stages, The discovered regularity in the formation of ordered supramolecular structure, depending upon ions and cell metabolites, plays a significant role in the control of skeletal muscle glycogenolysis in vertebrates. ACKNOWLEDGMENT. The authors wish to express their gratitude to Dr. B. I. Kurganov (A. N. Bach Institute of Biochemistry, Russian Academy of sciences, Moscow) for valuable discussions. REFERENCES 1. Clarke, F., Stephan, P., Morton, D., and Weidemann, J. (1985) In Regulation of cabrohydrate metabolism (Bietner, R., ed.), 2, pp. 1-31, CRC Press, New York 2. Kurganov, B.I. (1986) J. Theor. Biol., 119,

6 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL 3. Stere, P.A. (1987) Ann. Rev. Biochem., 56, Srere, P.A. (1985) Trends Biochem. Sci., 10, Meyer, F., Heilmeyer, L.G., Jr., Haschke, R.H., and Fischer, EH. (1970) J. Biol. Chem., 245, Klinov, S.V., Chebotareva, N.A., Lyssovskaya, N.P., Davidov, D.R., and Kurganov, B.I. (1982) Biochim. Biophys. Acta, 709, Kurganov, BI., Schors, EI., Klinov, S.V., and Chebotareva, N.A. (1995) Biochem. Mol. Biol. Intern., 36, Stura, E.A., Zanotti, G, Baru, Y.S., Sanson, M.S.P., Stuart, D.I., Wilson, K.S., Johnson, I.N., and Van de Werve, G. (1983) J. Mol. Biol., 170, Steiner, R.F., and Marshall, L. (1982) Biochim Biophys. Acta, 707, Zemskova, M.A., Shur, S.A., Skolysheva, L.K., and Vulfson, P.L. (1989) Biokhimiya, 54, (In Russian). 11. Krebs, E.G., Love, D.S., Bratvold, G.E., Trayser, K.A., Meyer, W.L., and Fischer, E.H. (1964) Biochemistry, 3, Andreeva, I.E., Livanova, N.B., Eronina, T.B., Silonova, GV., and Poglazov, B.F. (1986) Eur. J. Biochem., 158, Cohen, P. (1973) Eur. J. Biochem., 34, Morozov, V.E, Eronina, T.B., Andreeva, I.E., Silonova, G.V., Solovyeva N.V. Schors, E.I., Livanova, N.B., and Poglazov, B.F. (1989) Biokhimiya, 54, (In Russian). 15. Fischer, EH, and Krebs, E.G. (1958) J. Biol. Chem., 231, Serebrenikova, T.P. (1981) Zhurnal Evolutsionnoyi Biokhirniyi i Phyziologii, 27, (In Russian). 17. Shmelev, VK., and Serebrenikova, T.P. (1987) Ukrainsky Biokhimitcheskii Zhumal, 59, (In Russian). 18. Peterson, G.L. (I977) Anal. Biochem., 83, Serebrenikova, T.P., and ShmeIev, V.K. (1989) In Imemational symposium "Molecular organization of biological structures", abstract book 1, D 22, p Moscow 20. Tabatabai, L.B., and Graves, D.J. (1978) J. Biol. Chem., 253, Chan, K.-F.J., and Graves, D.J. (1982) J. Biol. Chem., 257,

Serrata) Alkaline Phosphatase

Vol. 41, No. 5, April 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 951-959 An Essential Tryptophan Residue of Green Crab (Syclla Serrata) Alkaline Phosphatase Wen-Zhu Zheng 1, Qing-Xi Chen