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In vitro studies of pecten maximus glycogen phosphorylase and the interconversion of their forms
C.mp. Bi.chcm. Phl~h,I.. V o l 62B. pp. 381 to 387. ~, PergamOll PI'css Ltd 1979 Printed in Oreal Blilaill
0305-0491 79 0401-0381502.(X)0
I N V I T R O STUDIES OF P E C T E N M A X I M U S G L Y C O G E N P H O S P H O R Y L A S E A N D THE I N T E R C O N V E R S I O N OF THEIR F O R M S ISABEL VAZQUEZ-BAANANTE and MANUEL ROSELL-PEREZbI~ School of Pharmacy, University of Barcelona, Barcelona-28, Spain (Receired I I July 1978) Abstract 1. In Pecten maximus the enzyme glycogen phosphorylase was found. Besides phosphorylase a and h, another inactive form, phosphorylase c appears to exist. Activation of phosphorylase c requires a concentration of 5'-AMP 20-fold higher than phosphorylase h. 2. Only phosphorylase a and h are found if organisms had suffered a certain degree of anoxia. 3. Preincubation of crude extracts wherein the glycogen phosphorylase was mainly present as c form caused the conversion: Phosph. c ---* Phosph. b --* Phosph. a. 4. The conversion from the inactive(s) phosphorylase to active form was catalysed by a kinase(s) enzyme. That conversion was gradual and inhibited by G-6-P. The sugar-phosphate did not inhibit the conversion of inactive phosphorylase c to phosphorylase h.
I NTRODUCTION There is a lack of information concerning the enzymes involved in glycogen metabolism in marine bivalves. However, it is well k n o w n that bivalves can support anaerobic conditions depending upon glycogen as energy source (De Z w a a n & Zandee, 1972). Glycogen phosphorylase (E.C. 2.4.1.1), an enzyme which catalyses glycogen degradation, exists in mammalian skeletal muscles in two forms. One, phosphorylase a, is active in the absence of Y-AMP, whereas the other, phosphorylase b, requires the nucleotide for activity (Fischer et al., 1970; Graves & Wang, 1972; Busby & Radda, 1976). In vitro interconversion of both forms occurred through phosphorylation and dephosphorylation reactions catalysed by protein kinase and protein phosphatase, respectively (Krebs & Fischer, 1956: Keller & Cori, 1955; Hurd et al.. 1966a). Glycogen phosphorylase h kinase catalyses the phosphorylase b to a conversion. This phosphorylation reaction involves the transfer of four phosphates from A T P to the serine groups in the subunits of the enzyme (Krebs & Fischer, 1956: Cohen, 1973; H a y a k a v a et al., 1973). The phosphorylase phosphatase(s)catalyses the a to h conversion, releasing inorganic phosphate from the enzyme (Hurd et al., 1966b). The glycogen phosphorylase and interconverting enzymes have been mainly studied in mammals. Only a few studies have included marine invertebrates (Cowgill & Cori, 1955; Cowgill, 1959; Shoger et al., 1973; Vfizquez et al., 1973; Vfi.zquez, 1975; Z a m m i t & Newsholme, 1976). Previously, we have established that the glycogen synthetase is present in the sea scallop (V~.zquezBaanante & RoselI-P6rez, 1978). We now report that glycogen phosphorylase exists in adductor muscle of Pecten maxirnus in two forms, phosphorylase a and Deceased January, 1977.
h. If organisms had no longer suffered anoxia, a form more inactive than phosphorylase b (phosphorylase c) was also found. An explanation as how the interconversion between these forms of glycogen phosphorylase takes place is offered. MATERIALS AND METHODS Condition ~f organisms The Pecten maximus organisms used in this study came from a culture park set up in "Ria de Arosa" (Galicia), from where they were periodically sent by air in optimum conditions to ensure survival (i.e. in poliexpan boxes wherein an elevated humidity and temperature of less than 10C was maintained). The bivalve muscles were utilized either on arrival at the laboratory, 10-15hr after being taken from the sea, or after having spent a period of adaptation 24, 48 72 or 88 hr after arrival, in an environment similar to their natural habitat (i.e. tanks of aerated sea water containing diatom and flagellate algae, in order to provide a convenient and adequate food supply), Organisms spending more than 15hr outside of their natural environment were considered as anoxic. The central adductor muscles used in the experiments were isolate from an average of 4-6 organisms for each assay. This tissue is characterized by its white color, vitreous appearance, striated fiber and rapid contraction (Grass6, 1960). It was used either immediately after dissection or after it had been frozen at - 2 0 C for no longer than 1 month. Glycooen phosphorylase measurements Central adductor muscle suspended (1:5 or 1:10 w/v) in 50raM Tris-HCl, 20mM EDTA, 50mM NaF and 10mM mercaptoethanol (pH 6.8) was homogenized in a motor driven Potter Elvehjem homogenizer with a teflon pestle. This and subsequent steps were carried out at 4'C. The homogenate was centrifuged at 6500 0 in a Sorvall RC2 for 20rain and then filtered through glass wool. The supernatant was referred to as "'crude extract" and it was used to assay glycogen phosphorylase. The crude extracts were recentrifuged at 90,000 0 for 60rain in an ultracentrifuge Beckman L2, and the supernatants obtained were used in glycogen phosphorylase kinetic studies. 381
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ISABEL VAZQUEZ-BAANANTEand MANUEl. ROSELL-Pf':REZ
One of the assays of phosphorylase activity was a modification of the method of Cori et al. (19551. The liberation of inorganic phosphate resulting from the incorporation of glucose-l-P into glycogen was determined. The mixture reaction contained 1 6 r a m G-l-P, 1",, glycogen, 5 0 m M Tris -HCI (pH 6.8~ and the enzyme source in a final volume of 100ld. The reaction was stopped by addition of 0.27",, a m m o n i u m molybdate in 0.54N SO,tH2 at O 4 C . The phosphate released was determined by the Fiske & Subbarrow (1925~ method. Phosphorylase activity was also followed as described by Gilboe et al. (1972). The reaction mixture contained 50 m M [l-~4C]glucosc-P (specific activity was 3000 5000 counts/min per llmolL 1% glycogen. 100 m M NaF, 50 m M fl-glyccrophosphatc (pH 6.8) and enzyme source. The reaction was stopped in a similar way as was described by T h o m a s et al. (1968} for glycogen synthetasc assa). In both methods the reaction mixtures were incubated, at 25 C, for I l Y l 5 m i n (Vhzquez, 1975), without 5'-AMP to assay phosphorylase a or in presence of 1 2 or 1 6 m M Y - A M P if assays of phosphorylase h or phosphorylase c were carried out.
5 m M ATP, 1 0 m M Mg 2+ were added to crude extracts and preincubated at 25 C. At various times aliquots of 30td were added to reaction mixtures to assay the a and (a + hl glycogen phosphorylase by Gilboe et al. (19721 method. The dilution of crude extracts and the high concentration of G - I - P in the reaction was enough to stop the phosphorylase kinase activity. Protein was determined b? thc method of Lowry et al. I1951 ). following the Ji (1973) modilication. The glycogen was assayed by the method of ('arroll t't aL (1956) using glucose as standard. Chem i('als
Glycogen from rabbit liver, glucose-6-P, glucose-l-P. ATP, A M P and EDTA were obtained from The Sigma Chemical Co. MgC12, NaF. Tris. fl-glycerophosphate and anthrone reactive were from Merck. and mercaptoethanol was from Eastman Kodak. The radioactive [l-I¢C]glucose-P has bccn obtained either from the New England Co. or the Radiochemical Center.
Phosphorylase kimlse
The muscle homogenate was prepared with 50raM Tris HC1, 5 m M EDTA (pH 7.8t. The activity was determined by the conversion from phosphorylase h to ~l when
R ES U LTS
In central a d d u c t o r m u s c l e from the sea scallop, g l y c o g e n p h o s p h o r y l a s e activity was f o u n d (V~izquez
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In ritro studies of Pecten maximus
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[G LU COGE NO]rng/m I Fig. 2. Effect of glycogen concentration on glycogen phosphorylase activity. The direct and reciprocal plots of velocity of glycogen phosphorylase vs were determined when the concentration of glycogen in the reaction mixtures was between 0 and 30mg/ml. The crude extracts were prepared as in Fig. 1. Glycogen phosphorylase activity was assayed in the absence (O) or in the presence of 1.5 mM 5'-AMP (o).
et al., 1973; Vfi.zquez, 1975; Zammit & Newsholme,
1976). As is shown in Fig. 1, the K,. of glycogen phosphorylase for G - I - P was 3.4mM in the presence of 1.5 mM 5'-AMP. The concentration of glycogen necessary to obtain 1.,/2 I/,,..... was 2.2 mg/ml. This value was not modified if 1.5 mM 5'-AMP was present; however, the V.,.... of the reaction was enhanced by its presence (Fig. 2). Glycogen phosphorylase activity is stimulated by 5'-AMP (Fischer et al., 1970). The presence of fwo binding sites for 5'-AMP per phosphorylase b dimer has been proposed by Merino et al. (1976). The effect of the nucleotide on phosphorylase activity from Pecten maximus was studied, and two different behaviours were observed if: (a) the muscles came from organisms kept in sea water until assays were run or from those kept out of their habitat less than 15 hr before experiments; (b) the organisms had suffered long anoxic periods (24-36 hr out of their natural habitat) before muscles were utilized in the preparation of the crude extracts. Two forms of glycogen phosphorylase, as in organisms with a greater degree of evolution, were found in the first condition. One of these forms, phosphorylase a had activity in absence of 5'-AMP; the other, phosphorylase h, requires 5'-AMP for activity. The enzyme was also found in a form more inactive than phosphorylase h, that we call phosphorylase c,
as in lobster (Cowgill, 1959). This latter form requires a high concentration of 5'-AMP for activity. Its K,,, value, 16 20raM, is about 20-fold higher than the K,, for phosphorylase h, 0.6-H mM (Fig. 3). When applying the second set of conditions, only phosphorylase a and h activities were found. The percentages of phosphorylase a were higher than in the former conditions and only the first plateau of Fig. 3 is observed {not illustrated). Cowgill & Cori (1955) have reported the presence of a form more inactive than phosphorylase h in lobster muscle. In Pecten maximus, gradual conversion from an inactive phosphorylase, phosphorylase c, into phosphorylase b was observed by preincubation of crude extracts. After 2 hr, total activation was achieved by 1 - 2 m M 5'-AMP, as in other muscular systems. Likewise, during this time of preincubation the 5'-AMP independent activity increased. The percentages of 3, 38 and 58°o of the a, b and c form, respectively, found at the initial point, changed into 33, 52 and (~87/o after 2 br of preincubation (Fig. 4). The assays consistently showed an enzyme conversion from a very inactive form (phosphorylase c) to less inactive form (phosphorylase b) to active form (phosphorylase a) in a sequence of reactions dependent on preincubation time. A rapid fall of phosphorylase a and a phosphorylase a to b conversion took place by preincubation
384
ISABEL VAZQUEZ-BAANANTEand MANUEL ROSELL-PEREZ
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[AMP] Fig. 3. Kinetic plot activation of glycogen phosphorylase by 5'-AMP. The crude extracts were obtained from organisms taken out of sea water just before the experiments were started or when they have spent less than 15 hr outside their natural habitat. The 5'-AMP concentration in the reaction mixture was between 0 and 2 0 m M . The K. value for phosphorylase b is represented by (O) and the K a for phosphorylase c by (O), v e r s i o n o f p h o s p h o r y l a s e a to b in t h e c r u d e extracts (Table li. P r e i n c u b a t i o n of t h e c r u d e extracts in t h e presence of A T P - M g 2+ c a u s e d a c o m p l e t e c o n v e r s i o n from
of crude extracts obtained from muscles having the enzyme activity mainly as 5'-AMP independent. The fluoride ion, a known inhibitor of the phosphatase enzyme (Keller & Cori, 1955), inhibited the con150-
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Fig. 4. Changes in glycogen phosphorylase activity by preincubation of crude extracts without any addition. Muscle from organisms which have not suffered anoxia were homogenized in 50raM Tris-HCl. 5 m M EDTA tpH 7.8). The crude extracts were preincubated at 25 C without additions. At different times of preincubation, aliquots were taken to assay the phosphorylase activity in the absence of 5'-AMP (F61) or in presence of 2 m M 5'-AMP ([]) or 1 6 m M 5'-AMP (1~).
In t'itro studies of Pecten maximus
Table 1. Phosphorylase a to phosphorylase b conversion Preincubation time (min) 0 25 55 125
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Table 2. Effect of G-6-P and Mg 2+ on phosphorylase kinase activity
% Phosphorylase a 50 100 ) NaF mM NaF mM NaF 100 74 52 35
100 94 81 65
Additions G-6-P Mg 2÷ (mM) (mM)
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The crude extracts and assays were followed as in Fig. 5. The phosphorylase a activity values observed after 90 rain of preincubation of the crude extracts added with 5 mM ATP, 10raM Mg 2÷ was considered 1007/,, of activity. The crude extracts were preincubated for 30 min previously to the ATP-Mg 2÷ additions.
5'-AMP dependent phosphorylase(s) to 5'-AMP independent form, suggesting the existence of phosphorylase kinase enzyme in Pecten m a x i m u s (Fig. 5). Both metabolites, Mg 2+ and ATP were necessary to activate the phosphorylase kinase (Vhzquez, 1975). The enzyme was not stimulated by U T P - M g 2 + ; however, the inactivation of glycogen phosphorylase was halted and the activity phosphatase was inhibited under these conditions (unpublished results). The conversion from inactive to active phosphorylase was interfered if A T P - M g 2÷ was added in presence of G-6-P (Krebs et al., 1964; Tu & Graves, 1973). Figure 5 shows the effect observed when crude extracts from Pecten m a x i m u s were preincubated with A T P - M g 2÷ without or with different amounts of G-6-P (2, 5 or 10mM). If G-6-P was absent, the almost absent glycogen phosphorylase activity was converted first of all to phosphorylase b and next to
2000
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10 10 10 10 25 45
2 5 10 5 5
Crude extracts obtained as in Fig. 5 were preincubated with or without NaF addition. The muscle utilized in these studies had been previously frozen at - 2 0 C . The percentages of phosphorylase a obtained in the absence of NaF or in its presence, previously to the preincubation, were considered to be 100% of activity.
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phosphorylase a and the conversion from b to a occurred. However, if G-6-P was also present during the preincubation, the b to a phosphorylase conversion was hindered, but little or no inhibition of the conversion from inactive phosphorylase to phosphorylase b was found even when G-6-P was at high concentration. It seems that the G-6-P only inhibited the b to a conversion. The percentages of inhibition increased markedly as G-6-P concentration rose (Table 2). No evidence was obtained that an increase in Mg 2+ concentration causes a decrease in inhibition caused by G-6-P as Tu & Graves (1973) indicated. On the contrary, as Table 2 shows, when the addition of Mg 2÷ was increased from 10 to 2 5 m M or 4 5 m M and G-6-P was at 5 m M , the inhibiting effect increased too.
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Fig. 5. Effect of G-6-P on phosphorylase kinase activity. Crude extracts obtained from frozen muscles homogenized, as previously mentioned in Material and Methods, were preincubated, at 25"C, without additions (O). Thirty min after its preincubation, different aliquots were added with: 5 mM ATP, I0 mM Mg z÷ without G-6-P (A) or when G-6-P was added in the amount of: ([2) 2mM+ ( I ) 5mM or (A) 10mM. Phosphorylase activity was assayed in the absence ( - - t or in the presence of 2mM 5'-AMP ( - - ) .
386
ISABEL V~.ZQUEZ-BAANANTEand MANUELROSELL-PEREZ DISCUSSION
In Pecten maximus, the enzyme glycogen phosphorylase appears in three forms with different requirements of 5'-AMP to exhibit their respective activities. This study has shown that organisms which have not suffered anoxic conditions for long, or have remained in their natural habitat until the experiments, the phosphorylase was mainly found as inactive form, phosphorylase c. However, anoxic conditions promoted an increase in the phosphorylase a in order to allow glycogen degradation. Our evidence of the increase in nucleotide-dependent glycogen phosphorylase activity, phosphorylase b, is based upon the fact that the most inactive phosphorylase, phosphorylase c, practically constant during the preincubation of the crude extracts, changed into phosphorylase h and then to phosphorylase a (Fig. 4). An increase in nucleotide dependent and independent 5'-AMP phosphorylase activity was also found in trout and sea urchin eggs (Yamamoto, 1968: Shoger et al., 1973) where glycogen phosphorylase is responsible for glycogen degradation. The in vitro activation of phosphorylase kinase by ATP-Mg 2+ promoted the conversion from inactive glycogen phosphorylase into active f o r m - i n crude extracts of Pecten maximus, The activity observed after phosphorylase kinase activation was higher than that found initially when measured in presence of 1-2 mM 5'-AMP. The conversion was a gradual procedure since a very inactive phosphorylase form changed first to phosphorylase h and subsequently to phosphorylase a (Fig. 5). G-6-P could separate the two-step conversion hindering the h to a conversion but not the inactive phosphorylase to phosphorylase h conversion. The G-6-P has an inhibitory effect on phosphorylase kinase catalysed reaction (Krebs et al., 1964}. Because phosphorylase h activity was inhibited by G-6-P (Battersby & Radda, 1976: Garcia, RosellP4rez & Vfizquez-Baanante, unpublished results) probably the former inhibition is due to the fact that the phosphorylase kinase, if only one, can not modify its own substrate, phosphorylase h. These results seem to be compatible with phosphorylation of glycogen phosphorylase through intermediate forms partially phosphorylated, as proposed by Hurd et al. (1966). In the sea scallop, conversion of glycogen phosphorylase involves at least a phosphorylase kinase enzyme responsible for the change of inactive glycogen phosphorylase form(s) into active form. On the other hand, a phosphorylase phosphatase, at least, would carry out the change of active into inactive phosphorylase form(s). The most inactive phosphorylase, phosphorylase c, would be completely dephosphorylated. It seems that phosphorylase c is, in Pecten maximus, a reserve of inactive phosphorylase to account for the less active form depending on the metabolic requirements for energy. However, at this time we can not come to any conclusion about its physiological implications, since the observed high requirement of 5'-AMP for activity in vitro were not in cells. There is little doubt that this molecular species is totally inactive under physiological conditions, A high content of G-6-P in cells, usually found under favorable food supply, would hinder the b to a phosphorylase
conversion even if ATP is high in the cell. This mechanism could prevent the futile glycogenolysis, and the synthesis of glycogen would start because G-6-P had been found to behave as glycogen synthetase phosphatase activator (Rosell-P~rez et al,, 1976) VfizquezBaanante & Rosell-P6rez, 1978). A phosphorylase form nonactivable by l - 2 m M Y-AMP was a l s o . observed in placenta (Augy & Cedar& 1976) and liver (Sutherland & Wosilait, 1956). Purification of glycogen phosphorylase from these bivalves, identification of its isoenzymatic composition and the knowledge of how phosphorylase b interacts with Y-AMP would help us to understand the mechanism of phosphorylase action. Acknowledgements This work was supported by "Beca de Formaci6n de Personal lnvestigador" from the Ministry of Education of Spain and a U.S. NSF grant GF-44115.
REFERENCES AUGUYA. & CEDARL. (1976) Activit~ glycog6ne phosphorylasique des placentas humains fi terme et immature. C.r. hehd. S~anc. Acad. Sci., Paris 283, 1515-1518. BATTERSBVM. K. & RADDAG. K. (1976) The stereospecificity of the glucose-6-phosphate binding site of glycogen phosphorylase h. FEBS Lett. 72, 319-322. BusBY S. J. W. & RADDA G. K. (1976) Regulation of the glycogen phosphorylase system from physical measurements to biological speculations. In Current Topics in Cellular Regulation, Vol. 10 (Edited by HORECKERB. L. & STADMANE. R.), pp. 89 160. Academic Press, New York. CARROLL N. V., LONGLEV R. W. & ROE J. H. (1956) The determination of glycogen in liver and muscle by use of the anthrone reagent. J. biol. Chem. 220, 583 593. COHEN P. (1973) The subunit structure of rabbit skeletal muscle phosphorylase kinase and the molecular basis of its activation reaction. Eur. J. Biochem. 34, 1-14. CORI G. T,. ILLIGWORTHB. & KELLER P. J. (1955) Muscle phosphorylase. In Methods in Enzymology (Edited by COLOWlCK S. P. & KAPLAN N. O.). Vol. I, pp. 200 205. Academic Press, New York. COWG1LL R. W. (19591 Lobster muscle phosphorylase: purification and properties, d. biol. Chem. 234, 314f~3153. COWGILE R. W. & COR1 C. F. (1955) The conversion of inactive phosphorylase to phosphorylase b and phosphorylase a in lobster muscle extract. J. biol. Chem. 216, 133-140. DE ZWAAN N. & ZANDEti D, I. (1972) the utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis, d. comp. Bioehem. Physiol. 43B, 47-54. FISCHER E. H., POCKERA. & SAARIJ. C. (1970) The structure, function and control of glycogen phosphorylase. In Essays in Biochemistry, Vol. 6 (Edited by CAMPBELL P. N. d~ DICKENS F.), pp. 23-69. Academic Press, New York. FISKE G. H. & SUBBAROWY. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. GILBOE D. P., LARSONK. L. & NUTTALF. L. (1972) Radioactive method for the assay of glycogen phosphorylase. Analvt. Biochem. 47, 20-27. GRASSY"P. P. (1960) Trait6 de Zoologic, Vol. 5, section 2, pp. 1884~1889. Masson, Paris. GRAVES D. J. & WANG J. H. (1972) z~-Glucan phosphorylase--chemical and physical basis of catalysis and regulation. In The Enzymes, Vol. 7 (Edited by BORER P.), pp. 435 466. Academic Press, New York. HAYAKAWAT., PERKINSJ. P., WALSH D. A. & KREBS E. G. (1973) Studies on the subunit structure of rabbit muscle phosphorylase kinase. Biochemistry 12, 574 580.
In ritro studies of Pecten maximus
HURl) S. S., NOVOA W. B.. HICKI!NBOTTOMJ. P. & FISCHER E. H. (1966a) Phosphorylase phosphatase from rabbit muscle. In Methods in Enzymoloyy, Vol. 8 (Edited by COLOWICK S. P. & KAPLAN N. 03, pp. 546-550. Academic Press, New York. Ht;RD S. S., TELLI!R D. & FISCHER E. H. (1966b) Probable formation of partially phosphorylated intermediates in the interconversion of phosphorylase a and b. Biochem. hiophys. Res. Commun. 24, 79 84. JI T. H. (1973) Interference by detergents, chelating agents and buffers with the Lowry protein determination. Analyt. Biochem. 52, 517 520. KELLER P. J. & CORI G. T. (19551 Purification and properties of the phosphorylase rupturing enzyme. J. biol. Chem. 214, 127 134, KREBS E. G. & FISCHER E. H. (1956) The phosphorylase h to a converting enzyme of rabbit muscle. Biochim. biophys. Acta 20, 150-157. KREBS E. G.. LOVE D. S., BRATVOLD G. E., TRAYSER K. A., MEYER W. L. & FISCHER E. H. (1964) Purification of rabbit skeletal muscle phosphorylase kinase. Biochenlistry 3, 1022 1033. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R, J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. MERINO C. G., GARCIA BLANC() F. & LAYNEZ J. (1976) Phosphorylase b tretamerization induced by AMP at 25 C. FEBS Lett. 67, 9- 12. ROSELL-P~_REZM., ITARTE E., GUmOVARXJ., VAZQUEZM. I. & CASTI~E1RAS M. J. (1976) Metabolic' Interc'onversion of Enzymes (Edited by SHALTIELS.), pp. 85-92. SpringerVerlag, New York. SHOGER R. L., ASAMI K., YASAMASU J. & FUJIWARA A, (1973) Activation of phosphorylase in sea urchin eggs by Ca -~+ and cyclic 3'5'-AMP. Expl Cell Res. 82, 375 382.
387
SUTHERLAND E, W. & WOS1LAITW. D. (1965) The relationship of epinephrine and glucagon to liver phosphorylase I. liver phosphorylase; preparation and properties. J. biol. Chem. 218, 45%468. THOMAS J. A., SCHLENDER K. K. & LARGER J. (1968) A rapid filter paper assay for UDPG-glucose-glycogen glucosyltransferase, including and improved biosynthesis of UDPG-14C-glucose. Anal l't. Bioehem. 25, 486-499. Tu J. I. & GRAVES D. J. (1973) Inhibition of the phosphorylase system from physical measurements to biological speculations. In Current Topics in Celhdar Regulation, Vol. 10 (Edited by HORECKER B. L. & STADMAN E. R.), pp. 89-160. Academic Press, New York. V,~ZQUEZ M, I., GARCIA-FERN,~NDEZ M. C. & ROSELLPI~REZ M. (1973) Enzymes of glycogen metabolism in shell-fish. XIII R6un. S.E.B. f157, Salamanca, Spain. V,~ZQUEZ-BAANANTEI. (1975) Estudio de los enzimas implicados en el metabolismo del glucogeno en mfisculo aductor de Pecten maximus. Ph.D. thesis, University of Barcelona. V,~ZQUEZ-BAANANTE I. & ROSI-LL-PI~REZM. (1979) Kinetics of the D and I glycogen synthetase forms and their interconversion in the sea scallop Pecten maximus. Submitted to Rev. esp. Fisiol. 35(1). YAMAMOTOM. (1968) Fish muscle glycogen phosphorylase. Can. J. Biochem. 46, 423-432. ZAMMIT V. A. & NEWSHOLME E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactato dehydrogenase, octopine dehydrogenase, phosphoenolpiruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxalacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Biochem, J. 160, 447 462.
0305-0491 79 0401-0381502.(X)0
I N V I T R O STUDIES OF P E C T E N M A X I M U S G L Y C O G E N P H O S P H O R Y L A S E A N D THE I N T E R C O N V E R S I O N OF THEIR F O R M S ISABEL VAZQUEZ-BAANANTE and MANUEL ROSELL-PEREZbI~ School of Pharmacy, University of Barcelona, Barcelona-28, Spain (Receired I I July 1978) Abstract 1. In Pecten maximus the enzyme glycogen phosphorylase was found. Besides phosphorylase a and h, another inactive form, phosphorylase c appears to exist. Activation of phosphorylase c requires a concentration of 5'-AMP 20-fold higher than phosphorylase h. 2. Only phosphorylase a and h are found if organisms had suffered a certain degree of anoxia. 3. Preincubation of crude extracts wherein the glycogen phosphorylase was mainly present as c form caused the conversion: Phosph. c ---* Phosph. b --* Phosph. a. 4. The conversion from the inactive(s) phosphorylase to active form was catalysed by a kinase(s) enzyme. That conversion was gradual and inhibited by G-6-P. The sugar-phosphate did not inhibit the conversion of inactive phosphorylase c to phosphorylase h.
I NTRODUCTION There is a lack of information concerning the enzymes involved in glycogen metabolism in marine bivalves. However, it is well k n o w n that bivalves can support anaerobic conditions depending upon glycogen as energy source (De Z w a a n & Zandee, 1972). Glycogen phosphorylase (E.C. 2.4.1.1), an enzyme which catalyses glycogen degradation, exists in mammalian skeletal muscles in two forms. One, phosphorylase a, is active in the absence of Y-AMP, whereas the other, phosphorylase b, requires the nucleotide for activity (Fischer et al., 1970; Graves & Wang, 1972; Busby & Radda, 1976). In vitro interconversion of both forms occurred through phosphorylation and dephosphorylation reactions catalysed by protein kinase and protein phosphatase, respectively (Krebs & Fischer, 1956: Keller & Cori, 1955; Hurd et al.. 1966a). Glycogen phosphorylase h kinase catalyses the phosphorylase b to a conversion. This phosphorylation reaction involves the transfer of four phosphates from A T P to the serine groups in the subunits of the enzyme (Krebs & Fischer, 1956: Cohen, 1973; H a y a k a v a et al., 1973). The phosphorylase phosphatase(s)catalyses the a to h conversion, releasing inorganic phosphate from the enzyme (Hurd et al., 1966b). The glycogen phosphorylase and interconverting enzymes have been mainly studied in mammals. Only a few studies have included marine invertebrates (Cowgill & Cori, 1955; Cowgill, 1959; Shoger et al., 1973; Vfizquez et al., 1973; Vfi.zquez, 1975; Z a m m i t & Newsholme, 1976). Previously, we have established that the glycogen synthetase is present in the sea scallop (V~.zquezBaanante & RoselI-P6rez, 1978). We now report that glycogen phosphorylase exists in adductor muscle of Pecten maxirnus in two forms, phosphorylase a and Deceased January, 1977.
h. If organisms had no longer suffered anoxia, a form more inactive than phosphorylase b (phosphorylase c) was also found. An explanation as how the interconversion between these forms of glycogen phosphorylase takes place is offered. MATERIALS AND METHODS Condition ~f organisms The Pecten maximus organisms used in this study came from a culture park set up in "Ria de Arosa" (Galicia), from where they were periodically sent by air in optimum conditions to ensure survival (i.e. in poliexpan boxes wherein an elevated humidity and temperature of less than 10C was maintained). The bivalve muscles were utilized either on arrival at the laboratory, 10-15hr after being taken from the sea, or after having spent a period of adaptation 24, 48 72 or 88 hr after arrival, in an environment similar to their natural habitat (i.e. tanks of aerated sea water containing diatom and flagellate algae, in order to provide a convenient and adequate food supply), Organisms spending more than 15hr outside of their natural environment were considered as anoxic. The central adductor muscles used in the experiments were isolate from an average of 4-6 organisms for each assay. This tissue is characterized by its white color, vitreous appearance, striated fiber and rapid contraction (Grass6, 1960). It was used either immediately after dissection or after it had been frozen at - 2 0 C for no longer than 1 month. Glycooen phosphorylase measurements Central adductor muscle suspended (1:5 or 1:10 w/v) in 50raM Tris-HCl, 20mM EDTA, 50mM NaF and 10mM mercaptoethanol (pH 6.8) was homogenized in a motor driven Potter Elvehjem homogenizer with a teflon pestle. This and subsequent steps were carried out at 4'C. The homogenate was centrifuged at 6500 0 in a Sorvall RC2 for 20rain and then filtered through glass wool. The supernatant was referred to as "'crude extract" and it was used to assay glycogen phosphorylase. The crude extracts were recentrifuged at 90,000 0 for 60rain in an ultracentrifuge Beckman L2, and the supernatants obtained were used in glycogen phosphorylase kinetic studies. 381
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ISABEL VAZQUEZ-BAANANTEand MANUEl. ROSELL-Pf':REZ
One of the assays of phosphorylase activity was a modification of the method of Cori et al. (19551. The liberation of inorganic phosphate resulting from the incorporation of glucose-l-P into glycogen was determined. The mixture reaction contained 1 6 r a m G-l-P, 1",, glycogen, 5 0 m M Tris -HCI (pH 6.8~ and the enzyme source in a final volume of 100ld. The reaction was stopped by addition of 0.27",, a m m o n i u m molybdate in 0.54N SO,tH2 at O 4 C . The phosphate released was determined by the Fiske & Subbarrow (1925~ method. Phosphorylase activity was also followed as described by Gilboe et al. (1972). The reaction mixture contained 50 m M [l-~4C]glucosc-P (specific activity was 3000 5000 counts/min per llmolL 1% glycogen. 100 m M NaF, 50 m M fl-glyccrophosphatc (pH 6.8) and enzyme source. The reaction was stopped in a similar way as was described by T h o m a s et al. (1968} for glycogen synthetasc assa). In both methods the reaction mixtures were incubated, at 25 C, for I l Y l 5 m i n (Vhzquez, 1975), without 5'-AMP to assay phosphorylase a or in presence of 1 2 or 1 6 m M Y - A M P if assays of phosphorylase h or phosphorylase c were carried out.
5 m M ATP, 1 0 m M Mg 2+ were added to crude extracts and preincubated at 25 C. At various times aliquots of 30td were added to reaction mixtures to assay the a and (a + hl glycogen phosphorylase by Gilboe et al. (19721 method. The dilution of crude extracts and the high concentration of G - I - P in the reaction was enough to stop the phosphorylase kinase activity. Protein was determined b? thc method of Lowry et al. I1951 ). following the Ji (1973) modilication. The glycogen was assayed by the method of ('arroll t't aL (1956) using glucose as standard. Chem i('als
Glycogen from rabbit liver, glucose-6-P, glucose-l-P. ATP, A M P and EDTA were obtained from The Sigma Chemical Co. MgC12, NaF. Tris. fl-glycerophosphate and anthrone reactive were from Merck. and mercaptoethanol was from Eastman Kodak. The radioactive [l-I¢C]glucose-P has bccn obtained either from the New England Co. or the Radiochemical Center.
Phosphorylase kimlse
The muscle homogenate was prepared with 50raM Tris HC1, 5 m M EDTA (pH 7.8t. The activity was determined by the conversion from phosphorylase h to ~l when
R ES U LTS
In central a d d u c t o r m u s c l e from the sea scallop, g l y c o g e n p h o s p h o r y l a s e activity was f o u n d (V~izquez
!ii j tG-I-pr' m°M' I0
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30
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[G-I-P],mM Fig. l. Direct and reciprocal plots of velocity of glycogen phosphorylase vs. The assays were conducted in presence of 1.5 m M 5'-AMP. G-1-P concentration in the reaction was between 0 and 50raM. The enzyme source was the 90,000 g supernatant fraction obtained from homogenates prepared with 50 m M Tris HCI, 5 m M EDTA, 5 0 m M N a F and 1 0 m M mercaptoethanol(pH6.81.
In ritro studies of Pecten maximus
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[G LU COGE NO]rng/m I Fig. 2. Effect of glycogen concentration on glycogen phosphorylase activity. The direct and reciprocal plots of velocity of glycogen phosphorylase vs were determined when the concentration of glycogen in the reaction mixtures was between 0 and 30mg/ml. The crude extracts were prepared as in Fig. 1. Glycogen phosphorylase activity was assayed in the absence (O) or in the presence of 1.5 mM 5'-AMP (o).
et al., 1973; Vfi.zquez, 1975; Zammit & Newsholme,
1976). As is shown in Fig. 1, the K,. of glycogen phosphorylase for G - I - P was 3.4mM in the presence of 1.5 mM 5'-AMP. The concentration of glycogen necessary to obtain 1.,/2 I/,,..... was 2.2 mg/ml. This value was not modified if 1.5 mM 5'-AMP was present; however, the V.,.... of the reaction was enhanced by its presence (Fig. 2). Glycogen phosphorylase activity is stimulated by 5'-AMP (Fischer et al., 1970). The presence of fwo binding sites for 5'-AMP per phosphorylase b dimer has been proposed by Merino et al. (1976). The effect of the nucleotide on phosphorylase activity from Pecten maximus was studied, and two different behaviours were observed if: (a) the muscles came from organisms kept in sea water until assays were run or from those kept out of their habitat less than 15 hr before experiments; (b) the organisms had suffered long anoxic periods (24-36 hr out of their natural habitat) before muscles were utilized in the preparation of the crude extracts. Two forms of glycogen phosphorylase, as in organisms with a greater degree of evolution, were found in the first condition. One of these forms, phosphorylase a had activity in absence of 5'-AMP; the other, phosphorylase h, requires 5'-AMP for activity. The enzyme was also found in a form more inactive than phosphorylase h, that we call phosphorylase c,
as in lobster (Cowgill, 1959). This latter form requires a high concentration of 5'-AMP for activity. Its K,,, value, 16 20raM, is about 20-fold higher than the K,, for phosphorylase h, 0.6-H mM (Fig. 3). When applying the second set of conditions, only phosphorylase a and h activities were found. The percentages of phosphorylase a were higher than in the former conditions and only the first plateau of Fig. 3 is observed {not illustrated). Cowgill & Cori (1955) have reported the presence of a form more inactive than phosphorylase h in lobster muscle. In Pecten maximus, gradual conversion from an inactive phosphorylase, phosphorylase c, into phosphorylase b was observed by preincubation of crude extracts. After 2 hr, total activation was achieved by 1 - 2 m M 5'-AMP, as in other muscular systems. Likewise, during this time of preincubation the 5'-AMP independent activity increased. The percentages of 3, 38 and 58°o of the a, b and c form, respectively, found at the initial point, changed into 33, 52 and (~87/o after 2 br of preincubation (Fig. 4). The assays consistently showed an enzyme conversion from a very inactive form (phosphorylase c) to less inactive form (phosphorylase b) to active form (phosphorylase a) in a sequence of reactions dependent on preincubation time. A rapid fall of phosphorylase a and a phosphorylase a to b conversion took place by preincubation
384
ISABEL VAZQUEZ-BAANANTEand MANUEL ROSELL-PEREZ
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[AMP] Fig. 3. Kinetic plot activation of glycogen phosphorylase by 5'-AMP. The crude extracts were obtained from organisms taken out of sea water just before the experiments were started or when they have spent less than 15 hr outside their natural habitat. The 5'-AMP concentration in the reaction mixture was between 0 and 2 0 m M . The K. value for phosphorylase b is represented by (O) and the K a for phosphorylase c by (O), v e r s i o n o f p h o s p h o r y l a s e a to b in t h e c r u d e extracts (Table li. P r e i n c u b a t i o n of t h e c r u d e extracts in t h e presence of A T P - M g 2+ c a u s e d a c o m p l e t e c o n v e r s i o n from
of crude extracts obtained from muscles having the enzyme activity mainly as 5'-AMP independent. The fluoride ion, a known inhibitor of the phosphatase enzyme (Keller & Cori, 1955), inhibited the con150-
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Fig. 4. Changes in glycogen phosphorylase activity by preincubation of crude extracts without any addition. Muscle from organisms which have not suffered anoxia were homogenized in 50raM Tris-HCl. 5 m M EDTA tpH 7.8). The crude extracts were preincubated at 25 C without additions. At different times of preincubation, aliquots were taken to assay the phosphorylase activity in the absence of 5'-AMP (F61) or in presence of 2 m M 5'-AMP ([]) or 1 6 m M 5'-AMP (1~).
In t'itro studies of Pecten maximus
Table 1. Phosphorylase a to phosphorylase b conversion Preincubation time (min) 0 25 55 125
(
Table 2. Effect of G-6-P and Mg 2+ on phosphorylase kinase activity
% Phosphorylase a 50 100 ) NaF mM NaF mM NaF 100 74 52 35
100 94 81 65
Additions G-6-P Mg 2÷ (mM) (mM)
100 100 93 86
18 48 91 54 100
The crude extracts and assays were followed as in Fig. 5. The phosphorylase a activity values observed after 90 rain of preincubation of the crude extracts added with 5 mM ATP, 10raM Mg 2÷ was considered 1007/,, of activity. The crude extracts were preincubated for 30 min previously to the ATP-Mg 2÷ additions.
5'-AMP dependent phosphorylase(s) to 5'-AMP independent form, suggesting the existence of phosphorylase kinase enzyme in Pecten m a x i m u s (Fig. 5). Both metabolites, Mg 2+ and ATP were necessary to activate the phosphorylase kinase (Vhzquez, 1975). The enzyme was not stimulated by U T P - M g 2 + ; however, the inactivation of glycogen phosphorylase was halted and the activity phosphatase was inhibited under these conditions (unpublished results). The conversion from inactive to active phosphorylase was interfered if A T P - M g 2÷ was added in presence of G-6-P (Krebs et al., 1964; Tu & Graves, 1973). Figure 5 shows the effect observed when crude extracts from Pecten m a x i m u s were preincubated with A T P - M g 2÷ without or with different amounts of G-6-P (2, 5 or 10mM). If G-6-P was absent, the almost absent glycogen phosphorylase activity was converted first of all to phosphorylase b and next to
2000
% inhibition of the b to a conversion
10 10 10 10 25 45
2 5 10 5 5
Crude extracts obtained as in Fig. 5 were preincubated with or without NaF addition. The muscle utilized in these studies had been previously frozen at - 2 0 C . The percentages of phosphorylase a obtained in the absence of NaF or in its presence, previously to the preincubation, were considered to be 100% of activity.
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385
phosphorylase a and the conversion from b to a occurred. However, if G-6-P was also present during the preincubation, the b to a phosphorylase conversion was hindered, but little or no inhibition of the conversion from inactive phosphorylase to phosphorylase b was found even when G-6-P was at high concentration. It seems that the G-6-P only inhibited the b to a conversion. The percentages of inhibition increased markedly as G-6-P concentration rose (Table 2). No evidence was obtained that an increase in Mg 2+ concentration causes a decrease in inhibition caused by G-6-P as Tu & Graves (1973) indicated. On the contrary, as Table 2 shows, when the addition of Mg 2÷ was increased from 10 to 2 5 m M or 4 5 m M and G-6-P was at 5 m M , the inhibiting effect increased too.
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Fig. 5. Effect of G-6-P on phosphorylase kinase activity. Crude extracts obtained from frozen muscles homogenized, as previously mentioned in Material and Methods, were preincubated, at 25"C, without additions (O). Thirty min after its preincubation, different aliquots were added with: 5 mM ATP, I0 mM Mg z÷ without G-6-P (A) or when G-6-P was added in the amount of: ([2) 2mM+ ( I ) 5mM or (A) 10mM. Phosphorylase activity was assayed in the absence ( - - t or in the presence of 2mM 5'-AMP ( - - ) .
386
ISABEL V~.ZQUEZ-BAANANTEand MANUELROSELL-PEREZ DISCUSSION
In Pecten maximus, the enzyme glycogen phosphorylase appears in three forms with different requirements of 5'-AMP to exhibit their respective activities. This study has shown that organisms which have not suffered anoxic conditions for long, or have remained in their natural habitat until the experiments, the phosphorylase was mainly found as inactive form, phosphorylase c. However, anoxic conditions promoted an increase in the phosphorylase a in order to allow glycogen degradation. Our evidence of the increase in nucleotide-dependent glycogen phosphorylase activity, phosphorylase b, is based upon the fact that the most inactive phosphorylase, phosphorylase c, practically constant during the preincubation of the crude extracts, changed into phosphorylase h and then to phosphorylase a (Fig. 4). An increase in nucleotide dependent and independent 5'-AMP phosphorylase activity was also found in trout and sea urchin eggs (Yamamoto, 1968: Shoger et al., 1973) where glycogen phosphorylase is responsible for glycogen degradation. The in vitro activation of phosphorylase kinase by ATP-Mg 2+ promoted the conversion from inactive glycogen phosphorylase into active f o r m - i n crude extracts of Pecten maximus, The activity observed after phosphorylase kinase activation was higher than that found initially when measured in presence of 1-2 mM 5'-AMP. The conversion was a gradual procedure since a very inactive phosphorylase form changed first to phosphorylase h and subsequently to phosphorylase a (Fig. 5). G-6-P could separate the two-step conversion hindering the h to a conversion but not the inactive phosphorylase to phosphorylase h conversion. The G-6-P has an inhibitory effect on phosphorylase kinase catalysed reaction (Krebs et al., 1964}. Because phosphorylase h activity was inhibited by G-6-P (Battersby & Radda, 1976: Garcia, RosellP4rez & Vfizquez-Baanante, unpublished results) probably the former inhibition is due to the fact that the phosphorylase kinase, if only one, can not modify its own substrate, phosphorylase h. These results seem to be compatible with phosphorylation of glycogen phosphorylase through intermediate forms partially phosphorylated, as proposed by Hurd et al. (1966). In the sea scallop, conversion of glycogen phosphorylase involves at least a phosphorylase kinase enzyme responsible for the change of inactive glycogen phosphorylase form(s) into active form. On the other hand, a phosphorylase phosphatase, at least, would carry out the change of active into inactive phosphorylase form(s). The most inactive phosphorylase, phosphorylase c, would be completely dephosphorylated. It seems that phosphorylase c is, in Pecten maximus, a reserve of inactive phosphorylase to account for the less active form depending on the metabolic requirements for energy. However, at this time we can not come to any conclusion about its physiological implications, since the observed high requirement of 5'-AMP for activity in vitro were not in cells. There is little doubt that this molecular species is totally inactive under physiological conditions, A high content of G-6-P in cells, usually found under favorable food supply, would hinder the b to a phosphorylase
conversion even if ATP is high in the cell. This mechanism could prevent the futile glycogenolysis, and the synthesis of glycogen would start because G-6-P had been found to behave as glycogen synthetase phosphatase activator (Rosell-P~rez et al,, 1976) VfizquezBaanante & Rosell-P6rez, 1978). A phosphorylase form nonactivable by l - 2 m M Y-AMP was a l s o . observed in placenta (Augy & Cedar& 1976) and liver (Sutherland & Wosilait, 1956). Purification of glycogen phosphorylase from these bivalves, identification of its isoenzymatic composition and the knowledge of how phosphorylase b interacts with Y-AMP would help us to understand the mechanism of phosphorylase action. Acknowledgements This work was supported by "Beca de Formaci6n de Personal lnvestigador" from the Ministry of Education of Spain and a U.S. NSF grant GF-44115.
REFERENCES AUGUYA. & CEDARL. (1976) Activit~ glycog6ne phosphorylasique des placentas humains fi terme et immature. C.r. hehd. S~anc. Acad. Sci., Paris 283, 1515-1518. BATTERSBVM. K. & RADDAG. K. (1976) The stereospecificity of the glucose-6-phosphate binding site of glycogen phosphorylase h. FEBS Lett. 72, 319-322. BusBY S. J. W. & RADDA G. K. (1976) Regulation of the glycogen phosphorylase system from physical measurements to biological speculations. In Current Topics in Cellular Regulation, Vol. 10 (Edited by HORECKERB. L. & STADMANE. R.), pp. 89 160. Academic Press, New York. CARROLL N. V., LONGLEV R. W. & ROE J. H. (1956) The determination of glycogen in liver and muscle by use of the anthrone reagent. J. biol. Chem. 220, 583 593. COHEN P. (1973) The subunit structure of rabbit skeletal muscle phosphorylase kinase and the molecular basis of its activation reaction. Eur. J. Biochem. 34, 1-14. CORI G. T,. ILLIGWORTHB. & KELLER P. J. (1955) Muscle phosphorylase. In Methods in Enzymology (Edited by COLOWlCK S. P. & KAPLAN N. O.). Vol. I, pp. 200 205. Academic Press, New York. COWG1LL R. W. (19591 Lobster muscle phosphorylase: purification and properties, d. biol. Chem. 234, 314f~3153. COWGILE R. W. & COR1 C. F. (1955) The conversion of inactive phosphorylase to phosphorylase b and phosphorylase a in lobster muscle extract. J. biol. Chem. 216, 133-140. DE ZWAAN N. & ZANDEti D, I. (1972) the utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis, d. comp. Bioehem. Physiol. 43B, 47-54. FISCHER E. H., POCKERA. & SAARIJ. C. (1970) The structure, function and control of glycogen phosphorylase. In Essays in Biochemistry, Vol. 6 (Edited by CAMPBELL P. N. d~ DICKENS F.), pp. 23-69. Academic Press, New York. FISKE G. H. & SUBBAROWY. (1925) The colorimetric determination of phosphorus. J. biol. Chem. 66, 375-400. GILBOE D. P., LARSONK. L. & NUTTALF. L. (1972) Radioactive method for the assay of glycogen phosphorylase. Analvt. Biochem. 47, 20-27. GRASSY"P. P. (1960) Trait6 de Zoologic, Vol. 5, section 2, pp. 1884~1889. Masson, Paris. GRAVES D. J. & WANG J. H. (1972) z~-Glucan phosphorylase--chemical and physical basis of catalysis and regulation. In The Enzymes, Vol. 7 (Edited by BORER P.), pp. 435 466. Academic Press, New York. HAYAKAWAT., PERKINSJ. P., WALSH D. A. & KREBS E. G. (1973) Studies on the subunit structure of rabbit muscle phosphorylase kinase. Biochemistry 12, 574 580.
In ritro studies of Pecten maximus
HURl) S. S., NOVOA W. B.. HICKI!NBOTTOMJ. P. & FISCHER E. H. (1966a) Phosphorylase phosphatase from rabbit muscle. In Methods in Enzymoloyy, Vol. 8 (Edited by COLOWICK S. P. & KAPLAN N. 03, pp. 546-550. Academic Press, New York. Ht;RD S. S., TELLI!R D. & FISCHER E. H. (1966b) Probable formation of partially phosphorylated intermediates in the interconversion of phosphorylase a and b. Biochem. hiophys. Res. Commun. 24, 79 84. JI T. H. (1973) Interference by detergents, chelating agents and buffers with the Lowry protein determination. Analyt. Biochem. 52, 517 520. KELLER P. J. & CORI G. T. (19551 Purification and properties of the phosphorylase rupturing enzyme. J. biol. Chem. 214, 127 134, KREBS E. G. & FISCHER E. H. (1956) The phosphorylase h to a converting enzyme of rabbit muscle. Biochim. biophys. Acta 20, 150-157. KREBS E. G.. LOVE D. S., BRATVOLD G. E., TRAYSER K. A., MEYER W. L. & FISCHER E. H. (1964) Purification of rabbit skeletal muscle phosphorylase kinase. Biochenlistry 3, 1022 1033. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R, J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. MERINO C. G., GARCIA BLANC() F. & LAYNEZ J. (1976) Phosphorylase b tretamerization induced by AMP at 25 C. FEBS Lett. 67, 9- 12. ROSELL-P~_REZM., ITARTE E., GUmOVARXJ., VAZQUEZM. I. & CASTI~E1RAS M. J. (1976) Metabolic' Interc'onversion of Enzymes (Edited by SHALTIELS.), pp. 85-92. SpringerVerlag, New York. SHOGER R. L., ASAMI K., YASAMASU J. & FUJIWARA A, (1973) Activation of phosphorylase in sea urchin eggs by Ca -~+ and cyclic 3'5'-AMP. Expl Cell Res. 82, 375 382.
387
SUTHERLAND E, W. & WOS1LAITW. D. (1965) The relationship of epinephrine and glucagon to liver phosphorylase I. liver phosphorylase; preparation and properties. J. biol. Chem. 218, 45%468. THOMAS J. A., SCHLENDER K. K. & LARGER J. (1968) A rapid filter paper assay for UDPG-glucose-glycogen glucosyltransferase, including and improved biosynthesis of UDPG-14C-glucose. Anal l't. Bioehem. 25, 486-499. Tu J. I. & GRAVES D. J. (1973) Inhibition of the phosphorylase system from physical measurements to biological speculations. In Current Topics in Celhdar Regulation, Vol. 10 (Edited by HORECKER B. L. & STADMAN E. R.), pp. 89-160. Academic Press, New York. V,~ZQUEZ M, I., GARCIA-FERN,~NDEZ M. C. & ROSELLPI~REZ M. (1973) Enzymes of glycogen metabolism in shell-fish. XIII R6un. S.E.B. f157, Salamanca, Spain. V,~ZQUEZ-BAANANTEI. (1975) Estudio de los enzimas implicados en el metabolismo del glucogeno en mfisculo aductor de Pecten maximus. Ph.D. thesis, University of Barcelona. V,~ZQUEZ-BAANANTE I. & ROSI-LL-PI~REZM. (1979) Kinetics of the D and I glycogen synthetase forms and their interconversion in the sea scallop Pecten maximus. Submitted to Rev. esp. Fisiol. 35(1). YAMAMOTOM. (1968) Fish muscle glycogen phosphorylase. Can. J. Biochem. 46, 423-432. ZAMMIT V. A. & NEWSHOLME E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactato dehydrogenase, octopine dehydrogenase, phosphoenolpiruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxalacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Biochem, J. 160, 447 462.