Dephosphorylationof Rabbit Skeletal Muscle Phosphorylase Kinase EVIDENCE AGAKNST THE OPERATION OF THE “SECOND-SITE PHOSPHORYLATION’ MECHANiSM OF REGULATION* (Received for publication, October 21, 1980, and in revised form, November 25, 1980)
Mahrukh K. Ganapathi, StevenR. Silberman, H. Paris, and ErnestY . C. Lee From the Departmentof Biochemistry, University of Miami Schoolof Medicine, Miami, Florida 33136
Thedephosphorylation of rabbitskeletalmuscle that of the a-subunit (7-9).Activation of the enzymeby phosphorylase kinase was studied using two purified CAMP-dependentprotein kinase was reported to be associated rabbit skeletal muscle protein phosphatases. The first only with phosphorylation of the P-subunit (9). Studies by enzyme (My = 32,000) corresponds to the form we have Hayakawa et al. (7,8) also showed a major correspondenceof previously termed protein phosphatase C. Phosphoryl- activation with phosphorylation of the P-subunit, although ase kinase was found to be rapidly dephosphorylated the correspondence was not complete. The phosphorylation by thisenzyme. The site ofdephosphorylationwas of the a-subunitposes an enigma, sinceit is apparently a silent examined,and it was shown that this enzymewas or gratuitous phosphorylation in terms of regulation by relatively specific for the dephosphorylationof the j3- CAMP-dependentprotein kinase. subunit phosphate,as compared to the a-subunit phosA role of the a-subunit phosphorylation was proposed by phate, of phosphorylasekinase.Phosphaterelease Cohen and Antoniw (12) in terms of the “second-site phosfrom the @-subunitwas approximately 100-fold faster than from the a-subunit. More importantly, dephos- phorylation” hypothesis. According to this hypothesis the asubunit phosphate has a regulatory function in dictating the phorylation of the fl-subunit phosphate was signifnot icantly affected by phosphorylation of the a-subunit. dephosphorylation of the ,&subunit. The cycle of events enThe dephosphorylation of phosphorylase kinase by a visaged is that phosphorylation of the P-subunit occurs fist second low molecular weight protein phosphatase, M, with concomitant activation of phosphorylase kinase, Only = 33,500, was also studied. The specific activity of this with the subsequent phosphorylation of the a-subunit is the enzyme toward phosphorylase kinase was only a frac-P-subunit amenable to dephosphorylation. Inessence, the tion of that exhibited by the M, = 32,000 phosphatase. control of the dephosphorylation of the P-subunit by a-subunit This enzyme removed phosphatefrom both the a- and phosphorylation requires a specitic property of the protein #-subunits but more rapidly (about4-fold) from the a- phosphatase involved in inactivating phosphorylase kinase, subunit. With neither of theseenzymepreparations i.e. P-phosphorylatedphosphorylase kinaseshould be dephoswas there any evidence for the regulationof fl-subunit phorylated at a much poorerrate (or not at all) by comparison dephosphorylationby phosphorylationof the a-subunit to a,P-phosphorylated phosphorylase kinase. as proposed byCohen and Antoniw((1973) FEBS Lett. In this report we describe a study of the dephosphorylation 34,43-47). of rabbit skeletal muscle phosphorylase kinase bytwo homogeneous protein phosphatase preparations isolated from rabbit skeletal muscle. Phosphorylase kinase (EC2.7.1.38) catalyzes the phosphorMATERIALS AND METHODS ylation of phosphorylase b and plays a key role in the reguPurification of Rabbit Skeletal Muscle Phosphatase-The two lation of glycogenolysis. Studies of the rabbit skeletal muscle enzyme have been of importance in the development of our low molecular weight phosphatases (phosphatase C-I and phosphacurrent concepts of protein phosphorylation as a regulatory tase C-It) were isolated from rabbit skeletal muscle (13).’ The phosphatase C-I preparation usedin the experiments described in mechanism (1-4).Discovered by Krebs and Fischer in 1956 this work had a specific activity of 9800 units/mg, and the phosphata% (5), phosphorylase kinase itself was found to be phosphory- C-I1 preparation had aspecific activity of 248 units/rng, both assayed lated by CAMP-dependent protein kinase (1-4, 6). Rabbit against phosphorylase a. Both enzyme preparations were apparently skeletal muscle phosphorylase kinase consistsof four subunit homogeneous by the criteria of sodium dodecyl sulfate disc gel electypes (7-9), a, B, y, and 8, ofM, = 145,000,128,000,45,000,and trophoresis. Assay of phosphorylase phosphatase activity was as 17,000, respectively (9, lo), and has a quaternary structure of previously described (14), one unit of enzyme activity being defined as that which converts 1nmol of phosphorylase a (dimer)/min. (
[email protected] addition, most muscle phosphorylase kinase prepPreparation of Rabbit SkeletalMuscle Phosphorylase Kinasearations contain minor amounts of a frfth subunit type, a’, Phosphorylase kinase was prepared from rabbit skeletal muscle acwhich represents the red muscle isozyme type (11). Both the cording to theprocedure described by Cohen (9),up to and including a- and P-subunits are phosphorylated by CAMP-dependent gel Ntration on Sepharose 4B. At this stage of purification, the protein kinase (7-9). The phosphorylation of the &subunit by phosphorylase kinase preparationshad specific activities of9-14 CAMP-dependent protein kinase is much more rapid than units/mg, assayed as described by Cohen (9) at pH 8.2 and activity ratios (pH 6.8h3.2) of less than 0.05. Following this the preparations
* This work was supported by Grant AM 18512 from the National Institutes of Health and in part by a grant from the American Heart Association, Central Florida Chapter, Inc., and Broward Chapter, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
were further purified on a DEAE-Sepharose column equilibrated with 50 m~ imidazole chloride, 2 m~ EDTA, I m M dithiothreitol, 20% glycerol, pH 7.2, using a linear NaCl gradient. Recoveries of about
60% of the applied protein were obtained in the latter stepand were
’ S . R. Silberman, H. Paris, and E. Y. C. Lee, manuscript in preparation.
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Dephosphorylation of Phosphorylase Kinase
3214
found to be dependent on the presence of glycerol in the buffers used. These preparations displayed only three major bands on sodium dodecyl sulfate disc gelelectrophoresis, corresponding to thea, /?,and y subunits. All preparations were found to contain an a' band. Preparations were stored at -20°C in 50mM imidazole, 2 mM EDTA, 1 mM dithiothreitol, 50% glycerol, pH 7.2. Phosphorylation of phosphorylase kinase was performed using a homogeneous preparation of the catalytic subunit of bovine heart CAMP-dependent protein kinase. The latterwas a geneous gift of Drs. D. L. Brautigan and E. H. Fischer of the University of Washington, Seattle. A typical reaction mixture contained 4 mg/ml of phosphorylase kinase, 40 pg/ml of protein kinase, 0.1 mM [y-""PIATP (600900 cpm/pmol),7 rn MgC12,50 mM imidazole chloride, 0.5 mM ethylene glycol bis(P-aminoethyl ether)N,N,N',N'-tetraacetic acid, 2 rn EDTA, 1 mM dithiothreitol, 50 m~ sodium fluoride, 20% glycerol, pH 7.2. Under these conditions "P-incorporation plateaued at an extent of about 2 mol of "P/mol of enzyme within 20 min of incubation at 30°C. In this paper we shall refer to the stoichiometry of phosphorylation in terms of the (aPyS) unit (M, = 335,000).Concentrations of phosphorylase kinase will also be expressed on this basis. For the preparation of phosphorylase kinase containing less than 1 mol of "2P/mol, a small excess (10-20%)over the calculated amounts of ATP required was added. Following the phosphorylation reactions, the preparations were cooled in ice and the excess ATP removed. This was accomplished by 2-3 treatments involving centrifugation through a bed of Sephadex G-50 (15) equilibrated with 50 mM imidazole chloride, 2 mM EDTA, 1 mM dithiothreitol, 0.1 M NaCl, 20% glycerol, pH 7.2 The phosphorylated enzyme was dialyzed against the same buffer containing 50% glycerol and stored at -20°C. Assay of Phosphorylase KinasePhosphatase-The reaction mixtures (0.05 m l ) contained 1 p~ "'P-labeled phosphorylase kinase, 1 m g / d of bovine serum albumin, 50 mM imidazole chloride, 2 mM EDTA, 1 mM dithiothreitol, pH 7.2. Following incubation at 30"C, the reactions were stopped by the additon of 10 pl of 50% trichloroacetic acid. The reaction mixtures were then centrifuged at 2000 X g for 10 min at 4°C. Twenty-five p1 of the supernatantwas then spotted onto filter paper circles and counted in a liquid scintillation counter. Activities were calculated on the basis that 1 unit of activity released 1 nmol of '+,/min. Sodium Dodecyl Sulfate Disc Gel Electrophoresis-This was performed as described by Weber and Osborn (16), except that the sodium phosphate buffer concentration was reduced to 50mM in the running buffer and 40 mM in the gels. Gels of 11 cm length and 5% acrylamide composition were generally used. The duration of electrophoresis was usually 8 h, at a current of 4 mA per tube. For the determination of the distribution of radioactivity between the a- and /?-subunits, the gels were sliced into 1-mm segments which were counted in 5 ml of Aquasol (New England Nuclear).
toward phosphorylase kinase. A preparation of phosphatase C-I, which had a specific activity of9800 units/mg toward phosphorylase a, was found to have a specific activity of 3700 units/mg toward phosphorylase kinase containing 2 mol of "P/mol. The time course of the reaction was found to be biphasic in experiments inwhich different amounts of protein phosphatase were added (Fig. 1). It is seen that 1 mol of phosphate/mol of phosphorylase kinase is rapidly removed, while the second molof phosphate is much more slowly removed. It was estimated that removal of the second mol of phosphate occurred at about 1/1OOth the rateof the removal of the first phosphate. In thesecond type of experiment which was performed, the reactions were stopped at various degrees of dephosphorylation, and thedistribution of radioactivity in the a and P bands was determinedafter sodium dodecyl sulfate disc gel electrophoresis. A typical result is shown in Fig. 2. The correspondence of the radioactivity with the a, a', and ,8 bands was c o n f i e d by comparison with gels stained with Coomassie blue. A similar distribution of "P-label for phosphorylated rabbit skeletal muscle phosphorylase kinase to thatshown in Fig. 2 has been reported by McCullough and Walsh (19). It is readily evident from the data shown in Fig. 2 that the /3-subunit phosphate is preferentially removed. Thus, phosphatase C-I is relatively specific for the dephosphorylation of the P-subunit phosphateand dephosphorylates the a-subunitvery poorly. The next question examined was whether phosphorylation of the a-subunit hadany influence on the dephosphorylation of the P-subunit. Phosphorylase kinase was phosphorylated to theextents of 0.6, 1.5, and 2.0 mol of "P/mol, respectively. These substratesrepresent ones in which the (a + a')$? ratios of incorporated "P were estimated by sodium dodecyl sulfate disc gel electrophoresis to correspond to values of0:1, 0.5:l and 1:1, respectively, within the experimental limitations of the technique. A typical result showing the initial rates of dephosphorylation of these substrates by protein phosphatase C-I is shown in Fig. 3. No major effect on the initial rates of dephosphorylation was observed. It was, therefore, concluded that thepresence of phosphate on the a-subunitdid not have a major effect on dephosphorylation of the P-subunit phosphate by protein phosphatase C-I. Dephosphorylation of Phosphorylase Kinase by Protein
RESULTS A N D DISCUSSION
Dephosphorylation of Phosphorylase Kinase by Protein Phosphatase C-I-The two protein phosphatases used were isolated after the ethanol precipitation procedure of Brandt et al. (17). This procedure has been shown to resultin the isolation of a M , 35,000 type of protein phosphatase (13, 14, 17, 18). The first enzyme had a molecular weight of 32,000 as determined by sodium dodecyl sulfate disc gel electrophoresis and was highly active toward phosphorylase a (8,000-12,000 units/mg of protein). This enzyme corresponds to the phosphorylase phosphatase first studied by us in rabbit liver and to which we have also given the name protein phosphatase C (for reviews see Refs. 13 and 18). The second enzyme was isolated as a protein of molecular weight of 33,500 as determined by sodium dodecyl sulfate disc gel electrophoresis and displayed a low specific activity toward phosphorylase a (250600 units/mg). A preliminary account of the purification and properties of these two enzymes is given in Ref. 13. In this work we will refer to the enzyme with a high specific activity toward phosphorylase a as protein phosphatase C-I and the low specific activity enzyme as phosphatase C-11. This terminology is used for convenience and not for the purpose of providing any generalizations regarding the classification of these proteins. Proteinphosphatase C-I was found to be highly active
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FIG. 1. Dephosphorylation of rabbit skeletal muscle phosphorylase kinase by protein phosphatase C-I. Rabbit muscle phosphorylase kinase containing 2 mol of 32P/molwas incubated with purified protein phosphatase C-I as described for the assay of phosphorylase kinase phosphatase under "Materials and Methods." Shown above is the time course of 32PL release when different amounts of phosphatase C-I were used. Concentrations of C-I used in the incubation mixtures were 0.008 pg/ml (O),0.08 pg/ml (A),and 1.6 pg/ ml respectively. Values of 32P,released are presented as moles of "P, released per mol of phosphorylase kinase. No release of 32P,was observed in theabsence of added phosphatase C-I.
(m),
Dephosphorylation of Phosphorylase Kinase
3215
phatase C-I1 toward phosphorylase kinase was extremely low. Values of 4-22 units/mg of protein were obtained using the same substrateswhich were usedto testthe activity of protein phosphatase C-I. The initial rates of dephosphorylation of phosphorylase kinase containing different amounts of phosphate by phosphatase C-I1 is shown in Fig. 4. It would appear from this data that there was an increase in initial rate with phosphorylation of the a-subunit. Thus, thereis an apparent effect of a-subunit phosphorylation in stimulating the dephosphorylation of the enzyme. Examination of the partially dephosphorylated phosphorylase kinase by sodium dodecyl sulfate disc gel electrophoresis revealed that it is, in fact, the a-subunit which is preferentially dephosphorylated (Fig. 5). The apparentstimulation of dephosphorylation with increasing a-subunit phosphorylation can be explained in terms of an increase inthe concentration of the preferred substrate andis not due to an enhancement of /3-subunit phosphate removal. Thus the phosphatase C-I1 enzyme preparation differs from the phosphatase C-I preparation in that it dephosphorylates the a-subunitpreferentially, and from the datain Fig. 4 it can be estimated that the a-subunit is dephosphorylated approximately four times faster than is the /3-subunit. It may be questioned as to whether the phosphatase C-I1 40 50 6040 50 60 activities are simply due to a cross-contamination by protein GEL SLICE NUMBER phosphatase C-I, since the latteris much more active than CFIG. 2. Subunit specificityof the dephosphorylation of a,/?- 11. This is patently not the case for the major activity of labeled phosphorylase kinase by protein phosphataseC-I. Rab- phosphatase C-11, which is toward the a-subunitphosphate of bit muscle phosphorylase kinase containing 2 mol of 32P/mol was incubated with purified protein phosphatase C-I (0.008 pg/ml of phosphorylase kinase. However, a very small contamination incubation mixture) as described under “Materials and Methods.” of phosphatase C-I1 by phosphatase C-I would be sufficient to The reactions were stopped by the addition of trichloroacetic acid account for the activity toward the /3-subunit phosphate disand the 32P, release determined as usual. In a duplicate set of tubes played by phosphatase c-11.However, it should be noted that the assays were terminated by heating at l00’C for 5 min. The the activity of phosphatase C-I was inhibited by the presence distribution of 32P-labelin the a- and P-subunitswas then determined of Mn”, whereas that of phosphatase C-I1 was stimulated. by sodium dodecyl sulfate disc gel electrophoresis as described under “Materials andMethods.” The distributions of ”P-label in the appro- When phosphorylase kinase phosphorylated only in the ppriate regions of the gels after 0’76,14%, 26%, and @%, of the total subunit was used as a substrate, the activity of phosphatase label had been released are shown in panels A , B , C, and D, respec- C-I1 was stimulated by the addition of Mn’+ cations, suggesttively. ing that thisactivity is not dueto thepresence of phosphatase
c-I.
Gratecos et al. (20, 31) have isolated phosphorylase phosphatase from rabbit skeletal muscle as a protein of M , 33,000 and have reported that their preparation did not dephosphorylate phosphorylase kinase in the absence of divalent cations. Their preparation is probably distinct from protein phosphatase C-I although it is possible that itmay be similar or identical with protein phosphatase (2-11.
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FIG. 3. The effect of a-subunit phosphorylation on the dephosphorylation of phosphorylase kinaseby protein phosphatase C-I. Rabbit skeletalmuscle phosphorylase kinase containing 0.6 (A),1.5 (O),and 2 (U)mol of 32P/mol, respectively, was incubated with protein phosphatase C-I (0.008 pg/ml of incubation mixture) as described under “Materials and Methods.” Phosphate release is expressed as moles of 32P,released per mol of phosphorylase kinase.
Phosphatase C-11-The protein phosphatase C-I1 preparation used for these studies had a specific activity of 248 units/ mg toward phosphorylase a. This preparation also dephosphorylated phosphorylase kinase. By comparison to protein phosphatase C-I (3000-4000 units/mg), the activity of phos-
-0
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10 TI ME (minutes)
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FIG. 4. The effect of a-subunit phosphorylation on the dephosphorylation of phosphorylase kinaseby protein phosphatase C-11. Rabbit skeletal muscle phosphorylase kinase containing 0.6 (A),1.5 (O),and 2 (B) mol of 32P/mol,respectively, was incubated with protein phosphatase C-I1 (0.52pg/ml of incubation mixture) as described under “Materials and Methods.” Phosphate release is expressed as moles of “‘P, released per mol of phosphorylase kinase.
Dephosphorylation of Phosphorylase Kinase
3216
ylation of glycogen synthase (22),lysine-rich histone (23),and pyruvate dehydrogenase (24). A corollary, but not a proof, of the second-site hypothesis is that one might expect the dephosphorylation of the a- and P-subunits to be carried out by distinct protein phosphatases. This expectation was confirmed by reports thatrabbit skeletal muscle contained distinctphosphatases with the required specificity. These were stated (25) to have “absolute specificity”althoughthey were studied only as partially purified preparations. The ‘‘,&phosphorylasekinase phosphatase” had a molecular weight of about 80,000 and was later correlated I with an enzyme fraction termed “protein phosphatase 111,” D which also dephosphorylated phosphorylase a and glycogen synthase (26). The a-phosphorylase kinase phosphatase was found to have a molecular weight of 180,000 and was later correlated withan enzyme fraction which was giventhe name “protein phsophatase 11” (26). In the work describing these preparations (25, 26), the question of the crucial property of the P-phosphatase, viz. the relative activity toward a$-phosphorylated and P-phosphorylated phosphorylase kinase in the absence of divalent cations, was not directly addressed. The studies presented in the paper are of considerable - 0 interest in regard to the “second-site phosphorylation” hy40 50 60 pothesis. Phosphatase C-I represents the major phosphorylase GEL SLICE NUMBER phosphatase activity present in the ethanol-treated muscle FIG. 5. Subunit specificity of the dephosphorylation of phos- extracts and is highly active toward phosphorylase kinase. phorylase kinase by protein phosphatase C-11. Rabbit skeletal Our findings with this enzyme are in complete disagreement muscle phosphorylase kinase containing 2 mol of ”P/mol was incubated with protein phosphataseC-I1 as described in the legend to Fig.with the operation of a second-site regulatory mechanism as originally proposed by Cohen (2,12). This can be categorically 4. Thedistribution of ”P-labelwasdetermined as described for phosphatase C-I in the legend to Fig. 2. Panels A , B , C, and D show stated in reference to thetwo enzyme preparations which we the distributionsof ‘’2P-labeled corresponding to the releaseof 0,1476, have studied. In preliminary studies we have examined the 19%, and 40%, respectively, of the total label. pattern of protein phosphatase activitiesin crude rabbit skeletal muscle extracts chromatographed on DEAE-Sepharose Dephosphorylation of Phosphorylase Kinase by an Endog- as described by Mellgren et al. (27). Using phosphorylase of 0.6 and 2.0 mol of ,‘y2P/ enous Phosphatase-The phosphorylase kinase preparations kinase phosphorylated to the extents used in these studies had been exposed to fluoride which was mol as the substrates, we have not been able to detect a removed before the enzyme was used. Under these conditions, phosphatase which dephosphorylates the ap-phosphorylated no detectable endogenous protein phosphatase activity was substrate but not theP-phosphorylated substrate. It has been shown that the a-subunit of phosphorylase noticed, provided that the reactions were carried out in the absence of divalent cations. If Mn2+was added, an appreciable kinase is phosphorylated much more slowly than the P-subrate of endogenous proteinphosphatase activity could be unit by CAMP-dependent protein kinase (7-9). Our in vitro measured. Examination of this Mn2’-stimulated activity in findings show that dephosphorylation of the a-subunitis also
z I C
terms of the pattern of dephosphorylation revealed that it preferentially dephosphorylates the a-subunit (Fig. 6) and resembles the phosphatase C-I1 activity. Relevance of the Current Findings to the Second-site Hypothesis-Evidence for the “second-site phosphorylation” hypothesis is based on a brief report by Cohen and Antoniw (12) in which the dephosphorylation of rabbitskeletal muscle phosphorylase kinase by endogenous phosphataseactivity was studied. In theabsence of divalent cations, removal of the P-subunitphosphate from the a$-phosphorylated enzyme was readily observed, whereas the ,&phosphorylated enzyme was very slowly dephosphorylated by comparison. On this basis they proposed that the role of the a-subunitphosphate was to regulate the dephosphorylation of the P-subunit phosphate. The crucial property of the P-subunit phosphatase which is necessary for the function of this proposed mechanism is that itdephosphorylates the @phosphorylated phosphorylase kinase much more rapidly than the P-phosphorylated enzyme. The only reported evidence available for this mechanism in relation to phosphorylase kinase is the original study dealing with the behavior of endogenous phosphatase(s) present as a contaminant in phosphorylase kinase preparations (12). The effects of second-site or multi-site phosphorylation of proteins on their dephosphorylation have also been examined in several other instances, uiz. for the dephosphor-
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GEL SLICE NUMBER FIG. 6. Dephosphorylation of phosphorylase kinase by an endogenous protein phosphatase. Rabbit skeletal muscle phosphorylase kinase was incubated as described for the phosphorylase kinase phosphatase assay (see under “Materials and Methods”) with the exception that no exogenous phosphatase was added, but with 5 mM MnC12 included in the assay. The distribution of ”P-label was determined after gel electrophoresis as described for phosphatase CI in the legend to Fig.2. Panels A, B, and C show the distributionsof 32P-label after release of 0, 8%. and 28%, respectively, of the total label.
Dephosphorylation of Phosphorylase Kinase a much slower process than that of the /?-subunit. In other words, there is a suggestion that theturnover of the a-subunit phosphate may occur at a much slower rate than thatof the P-subunit phosphate. This has obvious implications for the possible involvement of the a-subunit phosphate in the regulation of phosphorylase kinase activities by CAMP-dependent protein kinase-mediated reactions. Nevertheless, these considerations do not negate a role for a-subunit phosphorylation per se in the regulation of phosphorylase kinase activity. In vivo studies have shown that significant phosphorylation of the a-subunit occurs in both heart (19) and skeletal muscle (28). It is not completely agreed that phosphorylation of the a-subunit of rabbit skeletal muscle phosphorylase kinase by CAMP-dependent protein kinase does not contribute to activity change (7-9). A recent report indicates that phosphorylation of both a- and ,&subunits of bovine heart phosphorylase kinase can be correlated with activation of the enzyme (29). In addition, extensive phosphorylation of the &-subunit does occur in the autophosphorylation reaction catalyzed in the presence of calcium ions (1-4, 30). In this regard it should be noted that our studies have been carried out in the absence of divalent cations, and the possible influence of calcium ions, a physiologically important ligand of phosphorylase kinase (14) in modifying both the phosphorylation and dephosphorylation reactions, remains to be explored. The findings that rabbit skeletal muscle contains two low molecular weight phosphatases which are active on phosphorylase kinase has aparallel in the rabbitliver. It was originally reported by Khandelwal et al. (31) that two enzymesof M , 35,000 could beisolated to homogeneity fromthis tissue. Both enzymes were reported to dephosphorylate phosphorylase kinase, but the site specificity was not determined (31). A recent brief report by Ingebritsen et al. (32) provides supportive evicence that rat and rabbit liver both contain two M , 35,000 protein phosphatases, which display distinct specificities for the a- and /?-subunits of phosphorylase kinase in a similar fashion to that which we report here.
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Acknowledgments-We thank Dr. V. Dombradi for critical discussions of this work and Ms. G. G. Gutten for her technical assistance. REFERENCES 1. Carlson, G. M., Bechtel, P. J., and Graves, D. J. (1979) Adu. Enzymol. Relat. AreasMol. Biol. 50, 41-115 2. Cohen, P. (1978) Curr. Top. Cell. Regul. 14, 117-196 3. Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 48,
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923-959 4. Heilmeyer, L. M. G., Jr., Groschel-Stewart, U., Jahnke, U., Kilimann, M.W., Kohse, K.P.,and Varsanyi, M. (1980) Adv. Enzyme Regul. 18, 121-144 5. Krebs, E. G., and Fischer, E. H. (1956) Biochirn. Biophys. Acta 20,150-157 6. Walsh, D. A., Perkins, J. P.,and Krebs, E. G. (1968) J. Biol. Chem. 243,3763-3765 7. Hayakawa, T., Perkins, J. P., Walsh, D. A,, and Krebs, E. G. (1973) Biochemistry 12,567-573 8. Hayakawa, T., Perkins, J. P., and Krebs, E. G. (1973) Biochemistry 12,574-580 9. Cohen, P. (1973) Eur. J.Biochern. 34.1-14 10. Shenolikar, S.,Cohen, P. T. W., Cohen, P., Nairn, A. C., and Perry, S. V. (1979) Eur. J.Biochem. 100, 329-337 11. Jennissen, H. P., andHeilmeyer, L. M. G., Jr. (1974) FEBS Lett. 42,77-80 12. Cohen, P., and Antoniw, J. F. (1973) FEBS Lett. 3 4 , 4 3 4 7 13. Lee, E. Y. C., Silberman, S. R., Ganapathi, M. K., Petrovic, S., and Paris, H. (1980) Adu. Cyclic Nucleotide Res. 13, 95-131 14. Killiiea, S. D., Aylward, J. H., Mellgren, R. L., and Lee, E. Y. C. (1978) Arch. Biochem. Biophys. 191,638-646 15. Fry, D. W., White, J. C., and Goldman, I. D. (1978) Anal. Biochem. 90,809-815 16. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 17. Brandt, H., Capulong, Z. L., and Lee E. Y. C. (1975) J.Biol Chem. 250,8038-8044 18. Lee, E. Y. C., Mellgren, R. L., Killilea, S. D., and Aylward, J. H. (1978) FEBS Syrnp. 42, 327-346 19. McCullough, T. E., and Walsh, D. A. (1979) J. Biol. Chem. 254, 7345-7352 20. Gratecos, D., Detwiler, T. C., Hurd, S., and Fischer, E. H. (1977) Biochemistry 16,4812-4917 21. Detwiler, T. C., Gratecos, D., and Fischer, E. H. (1977) Biochemistry 16,4818-4823 22. Hutson, N. J., Khatra, B. S., and Soderling, T. R. (1978) J. Biol. Chem. 253,2540-2545 23. Usui, H., Nishimura, N., Imazu, M., Imaoka, T., Kinohara, N., and Takeda, M. (1979) Eur. J.Biochern. 99,413-417 24. Kerbey, A. L., and Randle, P. J. (1979) FEBS Lett. 108,485-488 25. Antoniw, J . F., and Cohen, P. (1976) Eur. J. Biochem. 68,45-54 26. Antoniw, J . F., Nimmo, H. G., Yeaman, S. J., andCohen, P. (1977) Biochem. J. 162,423-433 27. Mellgren, R. L., Aylward, J . H., Killilea, S. D., and Lee, E. Y. C. (1979) J.Biol. Chem. 254,648-652 28. Yeaman, S. J., and Cohen, P. (1975) Eur. J.Biochem. 51,93-104 29. Sul, H. S.,and Cooper, R. H. (1980) Fed. Proc. 39,2094 30. Wang, J . H., Stull, J . T., Huang, T. S., and Krebs, E. G. (1976) J. Biol. Chem. 251,4521-4527 31. Khandelwal, R. L., Vandenheede, J . R., and Krebs, E. G. (1976) J. Biol. Chem. 251,4850-4858 32. Ingebritsen, T. S., Foulkes, J. G., and Cohen, P. (1980) FEBS Lett. 119,9-15
