Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Guanosine 3',5'-monophosphate receptor protein: separation from adenosine 3',5'-monophosphate receptor protein.

A specific cGMP receptor protein has been identified and separated from the cAMP receptor protein by chromatography on 8-(6-aminohexyl)-amino-cAMP-Sepharose. Scatchard analysis of cGMP binding indicates a single affinity class of receptor sites with KD = 1.4 × 10^?8 M. The specificity of the cGMP receptor site has been defined by using a number of nucleotides as competitors for cGMP binding. The cGMP receptor protein sediments at 7S in glycerol density gradients.     

Functional specificity of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Adenosine 3':5'-monophosphate-dependent protein kinase partially purified from silkworm pupae shows identical functional activities with those of mammalian protein kinases; the insect and mammalian kinases are completely exchangeable in the phosphorylation of muscle glycogen phosphorylase kinase and glycogen synthetase resulting in the activation and inactivation of the respective enzymes. In contrast, guanosine 3':5'-monophosphate-dependent protein kinase obtained from the same organism is totally inactive in this role and phosphorylates different, mainly seryl and some threonyl, residues of acceptor proteins. Substrates of the latter kinase intimately involved in the regulation of biological processes have remained unknown.     

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.     

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.     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

An adenosine 3':5' monophosphate dependent protein kinase from sea urchin spermatozoa.

A cyclic AMP dependent protein kinase (EC 2.7.1.37) from sea urchin sperm as purified to near homogeneity and characterized. A 68-fold purification of the enzyme was obtained. This preparation had a specific activity of 389 000 units/mg protein with protamine as the substrate. On the basis of the purification required, it may be calculated that the protein kinase constitutes as much as 1.5% of the soluble protein in sperm. There appeared to be a single form of the enzyme in sea urchin sperm, based on the behavior of the enzyme during DEAE-cellulose and Sephadex G-200 column chromatography. Magnesium ion was required for enzyme activity. The rate of phosphorylation of protamine was stimulated 2.5-fold by an optimal concentration of 0.9 M NaCl. The Km for ATP (minus cyclic AMP) was 0.119 +/- 0.013 (S.D.) and 0.055 mM +/- 0.009 (S.D.) in the presence of cyclic AMP. The specificity of the enzyme toward protein acceptors, in decreasing order of phosphorylation, was found to be histone f1 protamine, histone f2b, histone f3 and histone f2a; casein and phosvitin were not phosphorylated. The holoenzyme was found to have an apparent molecular weight of 230 000 by Sephadex G-200 chromatography. In the presence of 5 - 10(-6) M cyclic AMP, the holoenzyme was dissociated on Sephadex G-200 to a regulatory subunit of molecular weight 165 000 and a catalytic subunit of Mr 73 000. The dissociation could also be demonstrated by disc gel electrophoresis in the presence and absence of cyclic AMP.     

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.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

Characterization and regulation of heart adenosine 3':5'-monophosphate-dependent protein kinase isozymes.

There is broad species variation in the type of cAMP-dependent protein kinase isozyme present in supernatant fractions of heart homogenates as determined by DEAE-cellulose chromatography, Isozyme I, which elutes at less than 0.1 M NaCl, is predominant in mouse and rat hearts; while isozyme II, which elutes at greater than 0.1 M NaCl, is the predominant type in beef and guinea pig. Human and rabbit hearts contain about equal amounts of the two types. The type I heart kinases are more easily dissociated into free regulatory and catalytic subunits by incubation with histone than are the type II kinases, and the separated regulatory and catalytic subunits of isozyme II of rat heart reassociate more rapidly than the subunits of isozyme I under the conditions used. The data from several experiments using rat heart indicate that the basal activity ratio of the protein kinase in crude extracts (approximately 0.15) is due mainly to basal endogenous cAMP and that cAMP elevation accounts entirely for the epinephrine effect on the enzyme. Addition of epinephrine and 1-methyl-3-isobutylxanthine to the perfusate causes a rapid (1 min) increase in cAMP, active supernatant protein kinase, and active phosphorylase in perfused hearts of both rat (mainly isozyme I) and guinea pig (mainly isozyme II). The elevation percentage in cAMP is about the same in the two species, but the increase in active protein kinase is greater in rat heart. If hearts from either animal are perfused continually (10 min) with epinephrine (0.8 muM) and 1-methyl-3-isobutylxanthine (10 muM), the cAMP level, active protein kinase, and active phosphorylase remain elevated. Likewise, all parameters return rapidly to the basal levels when epinephrine and 1-methyl-3-isobutylxanthin are removed. Most of the epinephrine effect on the rat heart supernatant kinase is retained at 0 degrees if cAMP is removed by Sephadex G-25 chromatography, although this procedure completely reverses the epinephrine effect in the guinea pig heart. The epinephrine effect on the rabbit heart kinase (approximately equal amounts of isozymes I and II) is partially reversed by Sephadex G-25. These species differences can be accounted for by differences in association-dissociation behavior of the isozymes in vitro. The data suggest that epinephrine causes activation of both isozymes. The activity present in the particulate fraction comprises nearly half of the total cAMP-dependent protein kinase activity in homogenates of rabbit heart. Triton X-100 extracts of low speed particulate fractions from hearts of each species tested, including rat heart, contain predominantly or entirely the type II isozyme, suggesting differences in intracellular distribution of the isozymes. The binding of the protein kinase to the particulate fraction is apparently due to the properties of the regulatory subunit component. Differences in topographical distribution of the isozymes could provide for differences in either physiological regulation or substrate specificity.     

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.