Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. I. Phosphorylation of histone fractions.

Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with H1, H2A, and H2B histones but not with H3 AND H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100 mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3',5'-monophosphate-dependent protein kinase.     

Isolation of stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian heart devoid of inhibitory modulator of adenosine 3':5'-monophosphate-dependent protein kinase.

The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.     

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.     

Partial purification and properties of guanosine 3':5'-monophosphate-dependent protein kinase from pig lung.

Guanosine 3':5'-monophosphate(cyclic GMP)-dependent protein kinase which catalyzes the phosphorylation of histone was purified about 200-fold from the soluble fraction of pig lung by pH 5.5 precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. The apparent Ka values for guanosine 3':5'-monophosphate and adenosine 3':5'-monophosphate were determined to be about 17 and 360 nM, respectively. Mg2+ was essential for the activity exhibiting biphasic stimulation behavior and neither Mn2+ nor Ca2+ could substitute for Mg2+. However, these divalent ions markedly inhibited the protein kinase activity stimulated by cyclic GMP in the presence of Mg2+.     

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.     

Comparison of mode of activation of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) partially purified from silkworm pupae was selectively activated by cyclic GMP at lower concentrations. Nevertheless, the enzyme seemed to differ from adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) with respect to the mode of response to cyclic nucleotides. The catalytic activity and cyclic GMP-binding activity were not dissociated by cyclic GMP in a manner similar to that described for protein kinase A. The enzyme was not inhibited by regulatory subunit of protein kinase A nor by protein inhibitor. A sulfhydryl compound such as 2-mercaptoethanol or glutathione was essential for the activation by cyclic GMP, and an extraordinary high concentration of either Mg2+ (100 mM) or Mn2+ (25 mM) was needed for maximal stimulation by cyclic GMP. A polyamine such as spermine, spermidine, or putrescine could substitute partly for the cation. Kinetic analysis indicated that Km for ATP was decreased whereas Ka for cyclic GMP was increased significantly at high concentrations of the cation. The effect of the cation to decrease Km for ATP was not evident in the absence of a sulfhydryl compound. These characteristics of protein kinase G described above were not observed for protein kinase A which was obtained from the same organism.     

Comparison of calcium-activated, cyclic nucleotide-independent protein kinase and adenosine 3':5'-monophosphate-dependent protein kinase as regards the ability to stimulate glycogen breakdown in vitro.

A cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka , Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.     

Adenosine 3':5'-monophosphate-regulated phosphorylation of erythrocyte membrane proteins. Separation of membrane-associated cyclic adenosine 3':5'-monophosphate-dependent protein kinase from its endogenous substrates.

An adenosine 3':5'-monophosphate (cyclic AMP)-binding protein in the human erythrocyte plasma membrane was isotopically labeled using a photoaffinity analog of cyclic AMP, N6-(ethyl 2-diazomalonyl) cyclic [3H]AMP. The cyclic AMP-binding site is located in a polypeptide chain having a molecular weight of 48,000. Cyclic AMP-binding protein and cyclic AMP-dependent protein kinase were solubilized with 0.5% Triton X-100 in 56 mM sodium borate, pH 8, but 32P-labeled membrane phosphoproteins were retained in the Triton-insoluble fraction, suggesting that the membrane-associated binding protein is not a primary substrate for protein kinase. Triton-solubilized and membrane-associated protein kinase activities were stimulated 15- and 17-fold by cyclic AMP, suggesting that the degree of association between the catalytic anc cyclic AMP-binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and a co-migrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.     

Guanosine 3':5'-monophosphate-dependent protein kinase from silkworm, properties of a catalytic fragment obtained by limited proteolysis.

Although guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) which was partially purified from silkworm pupae was not dissociated by cyclic GMP into catalytic and regulatory subunits as described for adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) (Takai, Y., Nakaya, S., Inoue, M., Kishimoto, A., Nishiyama, K., Yamamura, H., and Nishizuka, Y. (1976) J. Biol. Chem. 251, 1481-1487), limited proteolysis with trypsin resulted in the formation of catalytic and cyclic GMP-binding fragments which showed molecular weights of approximately 3.4 X 10(4) and 3.6 X 10(4), respectively (the molecular weight of native protein kinase G was 1.4 X 10(5)). The catalytic fragment did not bind cyclic GMP and was fully active in the absence of the cyclic nucleotide. The fragment did not show an absolute requirement for a sulfhydryl compound and high concentrations of Mg2+ (50 to 100 mM), both of which were necessary for the maximal activation of native protein kinase G. The catalytic fragment was not inhibited by the cyclic GMP-binding fragment nor by the regulatory subunit of protein kinase A. Inversely, the cyclic GMP-binding fragment was unable to inhibit the catalytic subunit of protein kinase A. Protein inhibitor, which was described for protein kinase A, was inert for the catalytic fragment.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

Purification of a protein inhibitor of adenosine 3':5'-monophosphate-dependent protein kinase from bovine myocardium by a non-denaturing procedure.

The activities of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase may be partially controlled by a ubiquitous acidic heat-stable protein which inhibits the phosphotransferase reaction by interaction with the catalytic subunit of protein kinase (Walsh, D.A. et al. (1971), J. Biol. Chem. 246, 1977-1985). Since reported purification of this inhibitor involved subjecting tissue extracts to denaturing conditions, its existence under physiological conditions remained uncertain. A protein inhibitor, molecular weight 22,500, has been isolated from bovine myocardium by methods that do not include exposure to extreme heat or acid precipitation. The activity of this acidic protein is destroyed by exposure to trypsin and is unaffected by treatment with neuraminidase, RNAse or DNAse.     

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.     

Inhibition of adenosine 3':5'-monophosphate-dependent protein kinase: comparison of a protein inhibitor with polyanions and substrate analogs.

The mechanism of inhibition of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase was studied using a protein inhibitor isolated by a non-denaturing procedure from bovine heart. This protein inhibitor interacts with the catalytic subunit of protein kinase and binds to some substrates of the kinase. Protein kinase activity can also be inhibited by polyanions which, like the protein inhibitor, bind to basic substrates but do not bind to the catalytic subunit of protein kinase. Peptides such as L-lysyl-L-tyrosyl-L-threonine that resemble the phosphate accepting site of protein kinase substrates competitively inhibit phosphorylation of histone. Protein kinase activity can thus be inhibited in vitro by interaction of the protein inhibitor with substrates, and/or the catalytic subunit of the kinase, by competition of substrate analogs with "natural" substrates and by direct interaction of polyanions with basic protein substrates for the phosphotransferase reaction.     

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.     

Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase.

Cardiac microsomes were incubated with [gamma-32P]ATP and a cardiac adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase in the presence of ethylene glycol bis(bets-aminoethyl ether)-N,N'-tetraacetic acid. After solubilization in sodium dodecyl sulfate and fractionation by polyacrylamide gel electrophoresis, a single microsomal protein component of approximately 22,000 daltons was found to bind most of the 32P label. The 32P labeling of this component increased several fold when NaF was included in the incubation medium. No other component of cardiac microsomes, including sarcoplasmic reticulum ATPase protein, contained significant amounts of 32P label. This 22,000-dalton phosphoprotein formed by cyclic AMP-dependent protein kinase had stability characteristics of a phosphoester rather than an acyl phosphate. Washing of microsomes with buffered KCl did not decrease the amount of 32P labeling to the 22,000-dalton protein, suggesting that this protein is associated with the membranes of sarcoplasmic reticulum rather than being a contaminant from other soluble proteins. The 22,000-dalton protein was susceptible to trypsin. Brief digestion with trypsin in the presence of 1 M sucrose did not significantly affect microsomal calcium transport activity, but prevented both subsequent phosphorylation of the 22,000-dalton protein and stimulation of calcium uptake by cyclic AMP-dependent protein kinase, suggesting that this protein is a modulator of the calcium pump. These results are consistent with previous findings (Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249, 6166-6173; Tada, M., Kirchberger, M.A., Repke, D.I., and Katz, A.M. (1974) J. Biol. Chem. 249, 6174-6180) that cyclic AMP-dependent protein kinase-catalyzed phosphorylation is associated with stimulation of calcium transport in the cardiac sarcoplasmic reticulum, and further indicate that this phosphorylation occurs at a component of low mass (22,000 daltons) of the cardiac sarcoplasmic reticulum which, while separable from the calcium transport ATPase protein (100,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has the ability to regulate calcium transport by the cardiac sarcoplasmic reticulum.     

Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles.

Effects of cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase were studied in sarcoplasmic reticulum prepared from cardiac and slow and fast (white) skeletal muscle. Cyclic AMP-dependent protein kinase failed to catalyze phosphorylation of fast skeletal muscle microsomes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyclic AMP-dependent protein kinase was without effect on calcium uptake by these microsomes. Treatment of cardiac microsomes obtained from dog, cat, rabbit, and guinea pig with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylation of a 22,000-dalton protein component in the amounts of 0.75, 0.25, 0.30, and 0.14 nmol of phosphorus/mg of microsomal protein, respectively. Calcium uptake by cardiac microsomes was stimulated 1.8- to 2.5-fold when microsomes were treated with cyclic AMP-dependent protein kinase. Protein kinases partially purified from bovine heart and rabbit skeletal muscle were both effective in mediating these effects on phosphorylation and calcium transport in dog cardiac sarcoplasmic reticulum. Slow skeletal muscle sarcoplasmic reticulum also contains a protein with a molecular weight of approximately 22,000 that can be phosphorylated by protein kinase. Phosphorylation of this component ranged from 0.005 to 0.016 nmol of phosphorous/mg of microsomal protein in dog biceps femoris. A statistically significant increase in calcium uptake by these membranes was produced by the protein kinase. Increases in protein kinase-catalyzed phosphorylation of a low molecular weight microsomal component and in calcium transport by sarcoplasmic reticulum of cardiac and slow skeletal muscle may be related to the relaxation-promoting effects of epinephrine seen in these types of muscle. Conversely, the absence of a relaxation-promoting effect of epinephrine in fast skeletal muscle may be associated with the lack of effect of cyclic AMP and protein kinase on calcium transport by the sarcoplasmic reticulum of this type of muscle.     

Stimulatory modulator of guanosine 3':5'-monophosphate-dependent protein kinase from mammalian tissues.

The crude protein kinase modulator preparations obtained from several rat tissues (aorta, brain heart, liver, lung, skeletal muscle, small intestine and testis) were separated into their stimulatory and inhibitory modulator components by Sephadex G-100 gel filtration. The isolated stimulatory modulator augmented the activity of guanosine 3':5'-monophosphate-dependent protein kinase. The isolated inhibitory modulator, on the other hand, depressed the activity of cyclic AMP-dependent protein kinase; it was without effect on the activity of cyclic GMP-dependent protein kinease. The present findings indicate that in the mammal, apparently in contrast to the arthropoda, separate proteins are responsibile for the stimulatory and the inhibitory activities of protein kinase modulator and that the two classes of cyclic nucleotide-dependent protein kinase are regulated in an opposing manner by these two types of modulators.     

Characterization of calf-ovary adenosine;3':5'-monophosphate-dependent protein kinases and adenosine-3':5'-monophosphate-binding proteins.

Protein phosphokinase activity from the calf ovary cytosol (105000 X g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major protein kinases, PK-H1 and PK-H2, both dependent on adenosine 3':5'-monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic-AMP-dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK-H1 of a unique cyclic-AMP-binding protein which has little catalytic activity associated with it. The cyclic-AMP-binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit. The ovarian cyclic-AMP-dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat-stable protein inhibitor isolated from rabbit skeletal muscle. Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic-AMP-dependent protein kinases and cyclic-AMP-binding proteins which include protein kinases and binding proteins of greater than 400 000 molecular weight.