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Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4843-4847, November 1977 Activation of hormone-sensitive and by purified cyclic GMP-dependent kinase (cyclic AMP-dependent /protein kinase inhibitor/cholesterol ) JOHN C. KHOO, PAMELA J. SPERRY, GORDON N. GILL, AND DANIEL STEINBERG Division of Metabolic Disease and Division of Endocrinology, Department of Medicine, University of California, San Diego, La Jolla, California 92093 Communicated by Nathan 0. Kaplan, August 12,1977

ABSTRACT Cyclic GMP-dependent protein kinase, purified The role of cAMP-dependent protein kinase in the activation to homogeneity from bovine lung, was shown to activate hor- of hormone-sensitive lipase from adipose tissue (9) and of mone-sensitive lipase partially purified from chicken adipose from (10) is well estab- tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher con- lished. The availability of a highly purified preparation of centrations of the cyclic GMP-dependent were required cGMP-dependent protein kinase (7) led- us to test the possibility (relative activities expressed in terms of histone H2b phospho- that this kinase might modify the activity of these intercon- rylation units). Activation by cyclic AMP-dependent protein vertible . kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the AND METHODS cyclic GMP enzyme was not inhibited. Lipase fully activated MATERIALS by cyclic AMP-dependent protein kinase showed no further Materials. ['4C]Triolein and cholesterol [1-14CJoleate were change in activity when treated with cyclic GMP-dependent purchased from Dhom Products, Ltd. Phosphorylase b (rabbit protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phos- skeletal muscle), phosphoglucomutase, -6-P dehydro- phorylase phosphatase (from bovine heart); full activity was genase, histone H2b, cAMP, cGMP, and ATP were obtained restored by reincubation with cyclic GMP and cyclic GMP- from Sigma Chemical Co. ['y-32P]ATP was prepared by the dependent protein kinase. Cholesterol esterase activity in the method of Glynn and Chappell (11). cAMP-dependent protein chicken adipose tissue fraction, previously shown to be activated kinase (specific activity, 45 nmol of 32p incorporated per mg along with the triglyceride lipase by cyclic AMP-dependent of histone H2b per mg of protein per min) was purified from protein kinase, was also activated by cyclic GMP-dependent to the method of Wastila et protein kinase. Crude preparations of hormone-sensitive tri- rabbit skeletal muscle according glyceride lipase from human or rat adipose tissue and choles- al. (12) through the first DEAE-cellulose chromatography step. terol esterase from rat adrenal were also activated by cyclic Protein kinase inhibitor was also purified from rabbit skeletal GMP-dependent protein kinase. Purified hosphorylase kinase muscle through the DEAE-cellulose chromatography step by (rabbit skeletal muscle) was also shown to be activated by cyclic the method of Walsh et al. (13). Phosphorylase kinase was pu- GMP-dependent protein kinase. The present results, together rified from rabbit skeletal muscle by the method of Cohen (14). with those of other workers on histone , suggest that the specificities of cyclic GMP-dependent and Phosphorylase phosphatase, a generous gift of E. Y. C. Lee, was cyclic AMP-dependent protein kinase may be similar. This is purified from bovine heart to homogeneity by-the method of discussed in the light of a model recently proposed with regard Brandt et al. (15). The specific activity was 7890 units/mg of to the relationship between the subunit structures of the two protein. . The physiologic significance of the findings remains Preparation of cGMP-Dependent Protein Kinase and to be established. Hormone-Sensitive Lipase. cGMP-dependent protein kinase was purified from bovine lung by affinity chromatography on Changes in intracellular concentrations of cyclic GMP (cGMP) 8-NH2(CH2)2NH-cAMP-Sepharose (7, 16). The homogeneous have been observed in association with a wide variety of met- enzyme prepared by elution with 0.1 mM cGMP in 2% Am- abolic and hormonal perturbations (reviewed in refs. 1 and 2). pholine and 10% glycerol was concentrated to 1 mg/ml. This In many circumstances the cellular levels of cGMP change purified enzyme had a specific activity of 1375 nmol of 32p reciprocally with those of cyclic AMP (cAMP), leading to the incorporated per mg of histone H2b/mg of protein per min. suggestion that these two cyclic regulate metabolic Because cGMP-dependent protein kinase was purified by processes in opposite directions-the yin-yang hypothesis (2). competitive elution with cGMP, removal of the by Under certain conditions, parallel changes in the concentrations chromatography on Sephadex G-200 or Sephadex G-50 at 300 of the two cyclic nucleotides are observed (3, 4). It has been was required when effects of added were to established in several mammalian systems that cAMP regulates be examined. enzymic activity by catalyzed by cAMP- Hormone-sensitive lipase from chicken adipose tissue was dependent protein kinase (5) and it has been suggested that prepared by the method of Khoo and Steinberg (17) to the pH cGMP may work in an analogous fashion through cGMP- 5.2 precipitate step. The specific activity ranged from 13 to 45 dependent protein kinase (6). The latter enzyme has now been nmol of oleic acid released per mg of protein per hr. Endoge- demonstrated in many tissues (cf. ref. 7) including adipose tissue nous cAMP-dependent protein kinase was inactivated by in- (8). Thus far, however, covalent enzyme modification with cubating the pH 5.2 precipitate fraction (5.2 P fraction) at 500 changes in enzyme activity catalyzed by cGMP-dependent for 20 min in the presence of 10 ,uM cAMP. This heat treatment kinase has not been demonstrated. caused a loss of 40% of the lipase activity. The cAMP was then The costs of publication of this article were defrayed in part by the removed by dialysis and chromatography on Sephadex G-50. payment of page charges. This article must therefore be hereby marked "adertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: cGMP, cyclic GMP; cAMP, cyclic AMP; EGTA, eth- this fact. ylene glycol-bis(,B-aminoethyl ether)-N,N'-tetraacetic acid. 4843 Downloaded by guest on October 1, 2021 4844 Biochemistry: Khoo et al. Proc. Nati. Acad. Sci. USA 74 (1977) Table 1. Activation of hormone-sensitive lipase from chicken adipose tissue* Lipase activity, Ratio: act. nmol oleic acid/ with additions/ mg protein act. with Additionst per hr MgATP alone MgATP alone 33 1.0 +cAMP 747 22.6 +cAMP and protein kinase inhibitor 41 1.2 +cGMP 30 0.9 +cGMP-dependent 0 10 20 30 40 50 protein kinase 72 2.2 Protein kinase, units/ml +cGMP and cGMP- FIG. 1. Activation of hormone-sensitive lipase as a function of dependent protein kinase 414 12.5 the concentration of cGMP-dependent protein kinase (0) and +cGMP, cGMP-dependent cAMP-dependent protein kinase (A). The chicken adipose tissue 5.2 protein kinase, and P lipase fraction (75 ,g) was first freed of endogenous cAMP-de- protein kinase inhibitor 515 15.6 pendent protein kinase by heat treatment in the presence of cAMP and then activated at 300 for 10 min under the conditions described * 5.2 P fraction (75 Mg) of chicken adipose tissue hormone-sensitive in Materials and Methods. In some cases, protein kinase inhibitor lipase was incubated with the indicated cofactors for 10 min at 300; (78 Mug/ml) was added to the incubation mixture (@, A). [14C]triolein emulsion was then added and incubation was continued for 30 min at 300. Lipase activity was determined from release of This heat treatment also inactivated endogenous phosphopro- [14C]oleic acid. tein phosphatase. t The concentrations of the cofactors were: Mg(OAc)2, 5 mM; ATP, 0.5 mM; cGMP or cAMP, 10,uM; protein kinase inhibitor, 78 Mug/ml; Enzyme Assay. The conditions for activation, deactivation, and cGMP-dependent protein kinase, 5.3 units/ml. and assay of hormone-sensitive lipase or cholesterol esterase were as described (18, 19). The activation mixture (0.1 ml) contained 5 mM magnesium acetate, 0.5 mM ATP, 10 ,uM alone. Addition of protein kinase inhibitor completely blocked cGMP or cAMP, cGMP- or cAMP-dependent protein kinase this, indicating that the activation was due to endogenous as indicated, 50-150 Mug of 5.2 P fraction, and 20% glycerol/i cAMP-dependent protein kinase. Addition of MgATP and mM EDTA/25 mM Tris, pH 7.4. After incubation at 300 for cGMP alone caused no activation, indicating that the endoge- 10 min, triglyceride lipase activity was assayed by adding 0.7 nous protein kinase of this adipose tissue fraction was not readily ml of an emulsion containing 0.1 mM [14C]triolein, bovine activated by cGMP under these conditions. This is in agreement serum albumin at 5 mg/ml, 2 mM EDTA, and 5 mM sodium with earlier studies in rat adipose tissue showing that, although (pH 7.0) and incubating for 30 min at 300. Free the Ka for cAMP-stimulated activation of hormone-sensitive [I4C]oleic acid was extracted with chloroform/methanol/ lipase was 1.1 X 10-7 M, significant cGMP-stimulated activa- benzene/water at pH 11.5 (19). Cholesterol esterase was assayed tion was seen only at 1 X 10-4 M (23). by using cholesterol [1-14C]oleate added in ethanol as described Addition of MgATP and cGMP-dependent protein kinase (18). yielded a 2-fold increase in triglyceride lipase activity; with Activation of phosphorylase kinase was carried out in a re- addition of both cGMP-dependent protein kinase and cGMP action mixture of 50 Mul containing 10 mM magnesium acetate, a 12-fold increase was obtained (Table 1). This approximately 0.3 mM ATP, 0.5 mM EGTA, 10MM cGMP or cAMP, cGMP- 6-fold enhancement by cGMP is comparable to the previously or cAMP-dependent protein kinase at the indicated concen- reported (7) enhancement by cGMP of cGMP-dependent trations, purified phosphorylase kinase at 0.25 mg/ml, 25 mM protein kinase activity in histone phosphorylation. Protein ki- f3-glycerophosphate, and 15 mM 2-mercaptoethanol, pH 6.8. nase inhibitor did not block activation due to cGMP-dependent After 20 min at 300, the reaction was terminated by addition protein kinase plus cGMP. On the contrary, it caused a slight of 0.5 ml of ice-cold buffer (25 mM ,B-glycerophosphate/15 mM increase (24%) in lipase activation, compatible with previous 2-mercaptoethanol, pH 6.8). Phosphorylase kinase was assayed reports that it enhances cGMP-dependent protein kinase acti- by a modification (20) of the method of Krebs et al. (21). vation (24). When both cAMP and cGMP (10-5 M) were added Phosphorylase a formed was assayed in the direction of glucose with cGMP-dependent protein kinase, there was no further 1-P formation (22). One unit of phosphorylase is defined as the increase in lipase activation above that seen with cAMP alone enzyme activity yielding 1 ,umol of glucose 1-P per min. (data not shown). cGMP-dependent protein kinase and cAMP-dependent In the course of these studies it was found that the endogenous protein kinase were assayed with histone H2b substrate as de- protein kinase associated with adipose tissue lipase could be scribed by Gill et al. (7). One unit of kinase activity is defined selectively and almost totally inactivated by heating in the as that amount of enzyme transferring 1 nmol of 32p from presence of cAMP, making activation dependent on addition [y-32P]ATP to recovered histone H2b per min. of cAMP-dependent protein kinase. Activation of the lipase as a function of added cAMP-dependent protein kinase is shown RESULTS in Fig. 1. Enzyme activity was increased more than 20-fold. Activation of Hormone-Sensitive Lipase from Chicken Addition of protein kinase inhibitor completely blocked this Adipose Tissue. The partially purified fraction of chicken activation. Substitution of purified cGMP-dependent protein hormone-sensitive lipase (5.2 P fraction) used in the first part kinase and cGMP under the same conditions yielded the same of these studies still contained significant levels of endogenous maximal degree of activation. Expressed in units based on cAMP-dependent protein kinase. Thus, as shown in Table 1, histone phosphorylation, cGMP-dependent protein kinase was activation was observed with addition of MgATP and cAMP less effective than cAMP-dependent protein kinase under the Downloaded by guest on October 1, 2021 Biochemistry: Khoo et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4845

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Cyclic nucleotide, M 0 10 20 30 FIG. 2. Effect of varying concentrations of cGMP (0) and cAMP Time, min (^)on the activation of hormone-sensitive lipase by cGMP-depen- FIG. 3. Reversible deactivation of hormone-sensitive lipase. dent protein kinase. Conditions for activation were as described in Chicken adipose tissue 5.2 P (750 ,g/ml) was fully activated with Materials and Methods except that 2 mM was included cGMP-dependent protein kinase. The activated enzyme preparation in the incubation mixtures and 100 jig of chicken adipose tissue 5.2 was immediately passed through a Sephadex G-50 column to remove P was used. The cGMP-dependent protein kinase preparation used ATP, cGMP, and Mg2+. The enzyme eluted in the void volume was wvas purified by affinity chromatography, and cGMP was removed supplemented with 5 mM Mg2+ and 0.35 unit of purified bovine heart by dialysis and chromatography on Sephadex G-200. The amount of phosphorylase phosphatase, and incubation was carried out at 300 cGMP-dependent protein kinase added was 7.8 units/ml. Basal lipase (0). Reactivation ofthe deactivated lipase was effected at 20 min by activity before the addition of cyclic nucleotides and cGMP-depen- ATP, 10 MM cGMP, and cGMP-dependent protein kinase at 5 dent protein kinase was 20 nmol of free fatty acid released per mg of units/ml and incubating for 10 min at 300 (t). The basal lipase ac- protein/hr. tivity prior to activation was 13 nmol of free fatty acid released per mg of protein/hr. conditions used. Addition of protein kinase inhibitor had no effect on the activation due to cGMP-dependent protein kinase, pation by endogenous cAMP-dependent protein kinase. The ruling out participation of cAMP-dependent protein kinase in degree of activation was comparable to that observed with the observed activation. cAMP-dependent protein kinase. Lipase activation by cGMP-dependent protein kinase as a Cholesterol esterase from rat adrenal has been previously function of cyclic nucleotide concentration is shown in Fig. 2. shown to be activated, although to a limited extent (40 + 16%), Half-maximal activation was obtained at X 10-7 M cGMP or by cAMP-dependent protein kinase (26, 27). As shown in Table 1.2 X 10-6 M cAMP, thus confirming the relative cyclic nu- 2, it was also activated by cGMP-dependent protein kinase. cleotide specificity of the cGMP-dependent protein kinase. Activation of Phosphorylase Kinase. Protein kinase-de- However, the Ka for cGMP and cAMP were about 10 times pendent activation of phosphorylase kinase (rabbit muscle) is greater than those reported for histone phosphorylation, and shown in Fig. 4. Activation by cAMP-dependent protein kinase the ratio cAMP/cGMP (i.e., 4:1) needed for half-maximal ac- plus cAMP was almost completely inhibited by added protein tivation was much lower than that reported for histone phos- kinase inhibitor. Full activation was also obtained with phorylation (ratio, 50:1) (7). Evidence that the activation by cGMP-dependent protein Table 2. Activation of hormone-sensitive lipase from human and kinase, like that by cAMP-dependent kinase, reflects protein rat adipose tissue and of cholesterol esterase from rat adrenal* phosphorylation of the lipase was obtained by demonstrating reversible deactivation by a purified (from Enzyme activity, bovine heart). As shown in Fig. 3, lipase previously activated nmol free fatty acid/mg protein per hr by cGMP-dependent protein kinase showed a progressive fall Human Rat Rat in activity during incubation with the phosphatase. At 20 mn. adipose adipose adrenal an aliquot was removed and again incubated (10 min) with tissue tissue cholesterol cGMP-dependent protein kinase and cGMP. This restored li- Additions lipase lipase esterase pase activity to that of the fully activated preparation. Mg2+ 118 44 30 Activation of Other Acyl . Previous studies have MgATP, cGMP 102 43 32 shown that activities against cholesterol and MgATP, cGMP, lower glycerides in chicken adipose tissue are closely associated cGMP-dependent with hormone-sensitive triglyceride lipase and, like it, are ac- protein kinase, tivated by cAMP-dependent protein kinase although to dif- protein kinase ferent degrees (18). In the present studies the cholesterol esterase inhibitor 289 70 42 and diglyceride hydrolase activities of the 5.2 P fractions were MgATP, cAMP, increased 5- to 6fold by cGMP-dependent protein kinase (data cAMP-dependent not shown). protein kinase 241 80 46 Hormone-sensitive triglyceride lipase in rat (23) and human (25) adipose tissue has been previously shown to be activated * The 5.2 P fraction ofhuman omental adipose tissue was used; in the by cAMP-dependent protein kinase. As shown in Table 2, both case ofthe rat tissues, the 100,000 X g supernatant fraction was used. are also activated The conditions for activation and the concentrations ofthe additions by cGMP-dependent protein kinase. Acti- were as described under Materials and Methods and in Table 1. The vation with cGMP-dependent protein kinase was carried out concentration of cAMP-dependent protein kinase used was 10 in the presence of protein kinase inhibitor to prevent partici- units/ml. Downloaded by guest on October 1, 2021 4846 Biochemistry: Khoo et-al. Proc. Natl. Acad. Sci. USA 74 (1977) did not observe activation of phosphorylase kinase by cGMP-dependent protein kinase. Although these authors used enzyme preparations isolated from other sources (silkworms or bovine cerebellum), it is unlikely that this explains the dif- ferent results because the cGMP-dependent protein kinases they studied phosphorylate histone at the same sites phosphorylated by cAMP-dependent protein kinase (31). Kuo et al. (35) also failed to obtain phosphorylase kinase activation with a 150- fold-purified cGMP-dependent protein kinase from fetal guinea pig lung, which would be expected to be very similar to the bovine lung enzyme used in the present studies. The possibility that other substances in partially purified preparations modify not only the level of activity but also substrate specificity was 0 2 4 6 8 10 considered. However, a partially purified preparation of Protein kinase, units/ml cGMP-dependent protein kinase, purified to the DEAE-cel- FIG. 4. Activation of phosphorylase kinase as a function of pro- lulose chromatography step prior to affinity chromatography tein' kinase concentration. The conditions for the activation of (7), catalyzed similar activation of hormone-sensitive lipase and phosphorylase kinase from rabbit skeletal muscle (12.5 ,tg) were as described under Materials and Methods. Activation by cAMP-de- phosphorylase kinase (data not shown). The use of higher pendent protein kinase plus cAMP was carried out either in the ab- concentrations of cGMP-dependent protein kinase (expressed sence (M) or in the presence (78 ,g/ml) of protein kinase inhibitor (A); as units of histone phosphorylating activity) and other differ- activation by cGMP-dependent protein kinase plus cGMP was carried ences in assay conditions may partially explain the difference out in the presence (78 ,ug/ml) of protein kinase inhibitor (0). The in results. activation was terminated by 1:10 dilution with ice-cold buffer, and Adipose tissue contains cGMP-dependent protein kinase phosphorylase kinase activity was immediately assayed as de- activity (8) and so changes in cGMP concentration might di- scribed. rectly regulate hormone-sensitive lipase. From the present re- sults it appears that regulation at this level would be in the same cGMP-dependent protein kinase but this required a somewhat direction as that exercised by cAMP. On the other hand, control higher concentration of kinase (expressed in histone phospho- of by cGMP might also be exercised at other levels and rylation units). This activation depended absolutely on the in an opposite direction to that exercised by cAMP. Illiano et presence of ATP. Activation by cGMP-dependent protein ki- al. (36) observed increases in cGMP in response to insulin, and nase was not inhibited by protein kinase inhibitor. In fact, the this has been confirmed by Fain and Butcher (37). However, data shown for cGMP-dependent protein kinase activation were the latter workers found little correlation between insulin- obtained in the presence of the same inhibitor concentration induced changes in cGMP levels and suppression of norepi- that fully inhibited cAMP-dependent protein kinase activa- nephrine-induced lipolysis. Carbachol also increases cGMP tion. levels but it does not appear to be antilipolytic (37). Further- more, itself has been reported to increase cGMP DISCUSSION levels, a finding difficult to reconcile with a simple push-pull Both cAMP- and cGMP-dependent protein kinases have been type of regulation. The levels of cGMP and cGMP-dependent purified to homogeneity (7, 28, 29). Although many physical protein kinase in rat adipose tissue are apparently low compared properties of the two enzymes are similar, the subunit structure to those of cAMP and cAMP-dependent protein kinase (8, 36, and response to cycle nucleotides differ. cAMP-dependent 37). Thus, it seems unlikely that under ordinary circumstances protein kinase is a tetramer, R2C2, which in the presence of the hormone-induced changes in cGMP levels would contribute cAMP exists as R2-(cAMP)2 + 2C (28-30). cGMP-dependent importantly to the lipolytic effect of . protein kinase is a dimer, (RC)2, which in the presence of cGMP Exton et al. (38) have reported that cGMP added to exists as (RC)2-(cGMP)2 (7, 16). perfusates increased phosphorylase activity. On the other hand, The present studies demonstrate the ability of purified in recent studies in heart and in liver, changes in cGMP con- cGMP-dependent protein kinase to act on two well-charac- centration have not been well correlated with changes in terized, interconvertible enzyme systems that have previously phosphorylase activity (4, 39). been shown to be activated by cAMP-dependent protein kinase Thus, it is difficult to assess the possible importance of (10, 23). Hormone-sensitive lipase activated by cGMP-depen- cGMP-dependent protein kinase in the activation of the lipase dent protein kinase was deactivated by a purified protein and phosphorylase kinase systems in vvo. The lesser potency phosphatase and then fully reactivated, supporting the inter- (based on histone phosphorylation units) of cGMP-dependent pretation that cGMP-dependent protein kinase catalyzes the protein kinase under the in vitro conditions used does not rule same phosphorylation as that catalyzed by cAMP-dependent out significant participation in vivo. cAMP- and cGMP-de- protein kinase (23). However, this remains to be demonstrated pendent protein kinases were compared on the basis of histone directly. Concurrently with the present studies, it was found phosphorylation because most published reports have utilized (D. K. Blumental, J. T. Stull, and G. N. Gill, unpublished data) this substrate. The homogeneous enzymes have similar specific that cGMP-dependent protein kinase also catalyzes the phos- activities: cAMP-dependent protein kinase, 1660 units/mg with phorylation of cardiac troponin. In these three cases, the reac- histone Type II A (28); cGMP-dependent protefinklnase, 1375 tions parallel those catalyzed in the same systems by cAMP- units/mg with histone H2b (7). Moreover, both protein kinases dependent protein kinase. Studies by Hashimoto et al. (31) have phosphorylate the same hydroxyl groups in histones H1 shown that the two kinases act on the same phosphorylation sites and H2b (31). Histone phosphorylation units are thus a rea- in histone. Taken together, the results suggest that the substrate sonable but arbitrary standard for comparison. specificities of the two kinases are similar. Whatever the physiologic significance, the finding of similar Nishiyama et al. (32), Takai et al. (33), and Inoue et al. (34) substrate specificities is consonant with the similarity in basic Downloaded by guest on October 1, 2021 Biochemistry: Khoo et al. Proc. Natl. Acad. Sci. USA 74 (1977) 4847 subunit structures in the model proposed by Gill (40). According 15. Brandt, H., Capulong, Z. L. & Lee, E. Y. C. (1975) J. Biol. Chem. to this model the two kinases may differ primarily in that the 250,8038-8044. regulatory and catalytic functions of cGMP-dependent protein 16. Gill, G. N., Walton, G. M. & Sperry, P. J. (1977) J. Biol. Chem. kinase are present in a single chain while in cAMP-dependent 252,6443-6449. 17. Khoo, J. C. & Steinberg, D. (1974) J. Res. 15, 602-610. protein kinase the chain is discontinuous-i.e., regulatory and 18. Khoo, J. C., Steinberg, D., Huang, J. J. & Vagelos, P. R. (1976) catalytic subunits are distinct. J. Biol. Chem. 251, 2882-2890. Note Added in Proof. A recent report by Lincoln and Corbin (41) 19. Pittman, R. C., Khoo, J. C. & Steinberg, D. (1975) J. Biol. Chem. documented that cGMP-dependent protein kinase catalyzed incor- 2,50,4505-4511. poration of 32P into rat liver and fructose-1,6-bis- 20. Khoo, J. C. (1976) Biochim. Biophys. Acta 422, 87-97. phosphatase, rabbit skeletal muscle synthase, and phospho- 21. Krebs, E. G., Love, D. S., Bratvold, G. E., Trayser, K. A., Meyer, rylase kinase, in agreement with the functional changes documented W. L. & Fischer, E. H. (1964) Biochemistry 3,1022-1033. in the present study. 22. Hardman, J. G., Mayer, S. E. & Clark, B. (1965) J. Pharmacol. Exp. Ther. 150,341-348. We thank Mrs. Mercedes Silvestre for her excellent technical assis- 23. Huttunen, J. K. & Steinberg, D. (1971) Biochim. Biophys. Acta tance. We are indebted to Dr. Ernest Y. C. Lee, University of Miami, 239,411-427. for the highly purified phosphorylase phosphatase, Dr. Steven E. Mayer 24. Donnelly, T. E., Jr., Kuo, J. F., Miyamoto, E. & Greengard, P. for the cAMP-dependent protein kinase, and Dr. Steven R. Gross for (1973) J. Biol. Chem. 248, 199-203. the phosphorylase kinase. This project was supported by National In- 25. Khoo, J. C., Aquino, A. A. & Steinberg, D. (1974) J. Clin. Invest. stitutes of Health Research Grant HL 12373 from the National Heart, 53, 1124-1131. Lung, and Blood Institute and Research Grant BC-209 from the 26. Trzeciak, W. H. & Boyd, G. S. (1974) Eur. J. Biochem. 46, American Cancer Society. G.N.G. is the recipient of Research Career 201-207. Development Award no. AM70215 from the National Institute of 27. Pittman, R. C. & Steinberg, D. (1977) Biochim. Biophys. Acta Arthritis, , and Digestive Diseases. 248,431-444. 28. Hofmann, F., Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1975) J. Biol. Chem. 250,7795-7801. 1. Goldberg, N. D., O'Dea, R. F. & Haddox, M. K. (1973) Adv. 29. Rubin, C. S., Erlichman, J. & Rosen, 0. M. (1972) J. Biol. Chem. Cyclic Nucleotide Res. 3, 155-223. 247,36-44. 2. Goldberg, N. D., Haddox, M. K., Nicol, S. E., Glass, D. B., San- 30. Rosen, 0. M. & Erlichman, J. (1975) J. Biol. Chem. 250, ford, C. H., Kuehl, F. A. & Estensen, R. (1975) Adv. Cyclic Nu- 7788-7794. cleotide Res. 5, 307-330. 31. Hashimoto, E., Takeda, M., Nishizuka, Y., Hamana, K. & Iwai, 3. Nesbitt, J. A., Anderson, W. B., Miller, Z., Pastan, I., Russell, T. K. (1976) J. Biol. Chem. 251, 6287-6293. & Gospodarowicz, D. (1976) J. Biol. Chem. 251, 2344-2352. 32. Nishiyama, K., Katakami, H., Yamamura, H., Takai, Y., Shi- 4. Pointer, R. H., Butcher, F. R. & Fain, J. N. (1976) J. Biol. Chem. momura, R. & Nishizuka, Y. (1975) J. Biol. Chem. 250, 1297- 251,2987-2992. 1300. 5. Krebs, E. G. (1972) Curr. Top. Cell. Regul. 5,99-133. 33. Takai, Y., Nishiyama, K., Yamamura, H. & Nishizuka, Y. (1975) 6. Kuo, J. F. & Greengard, P. (1970) J. Biol. Chem. 245, 2493- J. Biol. Chem. 250,4690-4695. 2498. 34. Inoue, M., Kishimoto, A., Takai, Y. & Nishizuka, Y. (1976) J. Biol. 7. Gill, G. N., Holdy, K. E., Walton, G. M. & Kanstein, C. B. (1976) Chem. 251, 4476-4478. Proc. Natl. Acad. Sci. USA 73,3918-3922. 35. Kuo. J. F., Kuo, W-N., Shoji, M., Davis, C. W., Seery, V. L. & 8. Lincoln, T. M., Hall, C. L., Park, C. R. & Corbin, J. D. (1976) Donnelly, T. E., Jr. (1976) J. Biol. Chem. 251, 1759-1766. Proc. Natl. Acad. Sci. USA 73,2559-2563. 36. Illiano, G., Tell, G. P. E., Siegel, M. I. & Cuatrecasas, P. (1973) 9. Steinberg, D. (1976) Adv. Cyclic Nucleotide Res. 7, 157-198. Proc. Natl. Acad. Scd. USA 70,2443-2447. 10. Walsh, D. A., Perkins, J. P. & Krebs, E. G. (1968) J. Biol. Chem. 37. Fain, J. N. & Butcher, F. R. (1976) J. Cyclic Nucleotide Res. 2, 243,3763-3765. 71-78. 11. Glynn, I. M. & Chappell, J. B. (1964) Biochem. J. 90, 147- 38. Exton, J. H., Hardman, J. G., Williams, T. F., Sutherland, E. W. 149. & Park, C. R. (1971) J. Biol. Chem. 246,2658-2664. 12. Wastila, W. B., Stull, J. T., Mayer, S. E. & Walsh, D. A. (1971) J. 39. Gardner, R. M. & Allen, D. 0. (1976) J. Pharmacol. Exp. Ther. Biol. Chem. 246,1996-2003. 198,412-419. 13. Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, 40. Gill, G. N. (1977) J. Cyclic Nucleotide Res. 3, 153-162. E. H. & Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985. 41. Lincoln, T. M. & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. USA 14. Cohen, P. (1973) Eur. J. Biochem. 34, 1-14. 74,3239-3243. Downloaded by guest on October 1, 2021