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J D Corbin

Publications and source records attributed to J D Corbin.

At least 91 records · Page 5Linked to original sources
PubMed1988

cAMP analogs used to study low-Km, hormone-sensitive phosphodiesterase.

The determination of cyclic nucleotide analog I50 values for phosphodiesterases is a relatively simple method to study interactions between the enzyme and cyclic nucleotide analogs. This approach allows a large number of derivatives to be tested for preliminary information concerning hydrolysis. To conclude that the I50 values is a measure of analog hydrolysis requires that the mechanism of inhibition of [3H]cAMP hydrolysis is competitive. It is possible that some analogs act as noncompetitive inhibitors. Provided the enzyme preparation is pure with respect to phosphodiesterases, the type of inhibition can be determined. When it is important to determine if an analog is hydrolyzed, the complementary method of measuring direct hydrolysis can be used. For the low-Km phosphodiesterase and the analogs studied here, relatively low I50 values are correlated with analog hydrolysis while relatively high I50 values are correlated with the absence of detectable hydrolysis. For analogs such as N6-benzoyl- and N6-monobutyryl-cAMP the method of determining I50 values provides information that is not obtainable by direct hydrolysis. For example, neither of these analogs appear to be hydrolyzed but N6-benzoyl-cAMP has a lower I50 value and therefore more readily interacts with the low-Km phosphodiesterase. This analog or other ones may be useful in cAMP analog affinity chromatography for purification of phosphodiesterases. The method for directly determining cAMP analog hydrolysis measures the disappearance of the substrate instead of appearance of the product. However, the method is very sensitive since some, but not all, cAMP analogs have lower activation constants than cAMP does. Therefore, analogs can be tested for hydrolysis at concentrations as low as 10-50 nM and small changes in analog concentration can be directed. The methods presented have not only provided information concerning the mechanisms and structural requirements for hydrolysis but the analog specificities for various phosphodiesterases can be used as one of the determinants of analog potency in intact cells. Furthermore, the correlation of analog I50 values as an indication of hydrolysis with the effects of insulin on analog-stimulated intact cell responses provides information concerning the mechanism of insulin action. Pitfalls. Since cAMP analog preparations may be contaminated with cAMP, it is advantageous to purify the analog before determining direct hydrolysis. A method using Sephadex G-25 is presented elsewhere in this volume.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

An assessment of phosphodiesterase activity in situ after treatment of hepatocytes with hormones.

The role of phosphodiesterase activation in controlling adenosine 3',5'-cyclic monophosphate (cAMP) levels within hepatocytes was investigated by preloading hepatocytes with the hydrolyzable cAMP analogue 8-para-chlorophenylthio-cAMP (8-pCl phi S-cAMP) and measuring disappearance of the analogue after treating the cells with various hormones. Incubation of hepatocytes with 15 nM 8-pCl phi S-cAMP increased the intracellular concentration of the analogue at 0.5 and 2 min, but by 5 min the concentration plateaued and remained constant or declined slightly at 7 and 10 min. Treatment of hepatocytes with 5 nM glucagon led to a rapid 50% decline in intracellular concentration of the analogue. However, 6 nM insulin produced no detectable change in analogue concentration, and a combination of 5 nM glucagon and 6 nM insulin produced no greater lowering of 8-pCl phi S-cAMP than did glucagon alone. Treatment of hepatocytes with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (50 microM) blocked approximately 30% of the glucagon-mediated decrease in 8-pCl phi S-cAMP concentration, and in separate cell incubations, it blocked 50% of the cAMP lowering produced by 125 nM 8-pCl phi S-cAMP. Treatment of analogue-preloaded hepatocytes with effective concentrations of phenylephrine, vasopressin, or angiotensin resulted in no change in intracellular analogue or cAMP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase.

Cyclic nucleotide analogs were used to study relaxation of pig coronary arteries and guinea pig tracheal smooth muscle in an attempt to determine the roles of cAMP- and cGMP-dependent protein kinases (cA-K and cG-K). In pig coronary artery strips, cGMP analogs were generally more effective than cAMP analogs in promoting relaxation of K+-induced contractions. Significant relaxation of this tissue was caused primarily by those cyclic nucleotide analogs that had high affinities for purified cG-K but not for cA-K. The low potencies of cA-K-specific analogs, as compared with cG-K-specific analogs, could not be readily explained by either unusually high susceptibilities to phosphodiesterases or low partition coefficients. The most potent cGMP analog, 8-(4-chlorophenylthio)-cGMP, exhibited a very slow reversibility of its relaxant effects in the intact tissue, consistent with its strong resistance to hydrolysis by phosphodiesterases measured in vitro. Pig coronaries contained atypically high levels of cGMP and cG-K, implying a potentially important role of this enzyme in smooth muscle function. Carbamylcholine-induced contractions of guinea pig tracheal segments were more sensitive than K+-induced pig coronary artery contractions to relaxation by cyclic nucleotide analogs. Consequently, the number of analogs that could be studied was significantly expanded. The cGMP analogs were again generally more potent, and the effectiveness of both cGMP and cAMP analogs in relaxing this preparation correlated with the Ka of the analogs for in vitro activation of cG-K, but not cA-K. A particularly strong correlation was observed when the effects of analogs modified only at the C-8 position were examined. A known target enzyme of cA-K, phosphorylase, was not activated by cG-K-specific analogs but was activated by high concentrations of the cA-K-specific analogs. Studies using cyclic nucleotide analogs support a role for cG-K, but not for cA-K, in decreasing smooth muscle tone.

Animals↗
PubMed1987

Interconvertible cGMP-free and cGMP-bound forms of cGMP-dependent protein kinase in mammalian tissues.

Several vascular and nonvascular mammalian tissue extracts exhibited variable amounts of two peaks (peaks I and II) of cGMP-dependent protein kinase by NaCl elution of DEAE columns. When [3H]cGMP was added to the extracts before chromatography, a peak of protein-bound [3H]cGMP coeluted with peak II. [3H]cGMP was added to purified bovine lung cyclic nucleotide-free enzyme followed by chromatography on high performance liquid chromatography-DEAE. Two kinase peaks, the first of which represented mainly cGMP-free enzyme and the second of which represented cGMP-bound enzyme, eluted at the same positions as peaks I and II, respectively, of the crude extracts. The relative amount of peak II increased as a function of increasing the [3H]cGMP added before chromatography, and peak II could be converted partially to peak I by rechromatography. The holoenzyme is known to contain two slowly exchanging cGMP binding sites (sites 1) and two rapidly exchanging sites (sites 2). Some protein-bound [3H] cGMP found entirely in site 1 coeluted with peak I, although most of the enzyme in that peak was cGMP-free. When low [3H]cGMP was used for the initial incubation, relatively more of the protein-bound [3H] cGMP appeared in peak I and could represent binding of [3H]cGMP to only one of the two sites 1 of the kinase. The [3H]cGMP bound to the peak II enzyme completely filled both sites 1. Cyclic GMP binding to these sites caused the apparent conformational change which shifted the DEAE elution position of the enzyme. The peak II kinase was partially active and had a higher sensitivity to further cGMP activation of kinase than did the cGMP-free enzyme, suggesting that activation of kinase by binding of cGMP to site 2 was facilitated by prior binding at site 1. In fractions of the trailing edge of peak II, the kinase activity was virtually cGMP-independent, and both sites 1 and 2 were almost saturated with [3H]cGMP. These results suggested a further conformational change and direct increase in activity by binding of cGMP at site 2.

Animals↗
PubMed1987

Purification and characterization of an inactive form of cAMP-dependent protein kinase containing bound cAMP.

By a new procedure, the holoenzyme of bovine heart type II cAMP-dependent protein kinase was purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A high performance liquid chromatography-DEAE purification step resolved two distinct peaks of protein kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. The two peaks exhibited similar Stokes radii and sedimentation coefficients. They had similar ratios of regulatory to catalytic subunits both by densitometric scanning of SDS-PAGE bands and by the ratios of equilibrium [3H]cAMP binding to maximal kinase activity. These results suggested that the holoenzyme of each peak contained two regulatory subunits and two catalytic subunits, although a subpopulation of holoenzyme lacking one catalytic subunit also appeared to be present in Peak 2. Assays of cAMP indicated that the Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Determination of [3H]cAMP dissociation rates showed that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP. Interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2. Peak 2 holoenzyme, as compared to Peak 1, had enhanced binding in nonequilibrium [3H]cIMP and [3H]cAMP binding assays, as was expected due to the presence of cAMP and to the known positive cooperativity in binding of cyclic nucleotides to the kinase. The positive cooperativity in kinase activation, as indicated by the Hill coefficient, was greater for Peak 2 than Peak 1, but the cAMP concentration required for half-maximal activation (Ka) of each of the two peaks was very similar. In conclusion, Peak 2 is an inactive ternary complex of cAMP, regulatory subunit, and catalytic subunit, and Peak 1 is a cAMP-free holoenzyme. The cAMP-bound form may represent a major cellular form of the enzyme which is primed for activation.

Animals↗
PubMed1987

Studies on the interactions between the cyclic nucleotide-binding sites of cGMP-dependent protein kinase.

The rate and equilibrium kinetics of [3H]cGMP binding to the two rapidly exchanging and two slowly exchanging sites of dimeric cGMP-dependent protein kinase from bovine lung were studied. As observed by McCune and Gill (McCune, R. W., and Gill, G. N. (1979) J. Biol. Chem. 254, 5083-5091), unlabeled cGMP retarded the dissociation of [3H]cGMP bound to the "slow" site. This effect was due to interaction of unlabeled cGMP with the "rapid" rather than the slow site. First, the potencies of unlabeled cGMP and a number of cGMP analogs correlated nearly perfectly with their affinities for the rapid site. Second, the rate of dissociation in the absence of unlabeled ligand was independent of the degree of saturation of the slow sites. Third, unlabeled ligand inhibited the rate of dissociation more (about 10-fold) than theoretically predicted (maximum 2-fold) from interaction between two similar sites in one macromolecule. A favorable free energy coupling appeared to exist between the rapid and slow sites but not between the slow sites. cGMP associated faster to the slow site than the rapid site. Mg/ATP decreased the rate of association to either site by 50% and increased about ten-fold the rate of dissociation from the slow site. The dissociation of cGMP from the slow site could be described by a single activation energy (Ea = 71 kJ X mol-1) for the whole temperature range (0-37 degrees C) tested. These data indicated that the cyclic nucleotide-binding sites of the cGMP-kinase are kinetically more homologous to those in the cAMP-dependent protein kinases than previously recognized.

Animals↗
PubMed1987

Short-term feedback regulation of cAMP by accelerated degradation in rat tissues.

A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes (Corbin, J.D., Beebe, S.J., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 8731-8735). The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pCl phi S-cAMP (where 8-pCl phi S- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pCl phi S-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pCl phi S-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pCl phi S-cAMP occurred in parallel with increases in lipolysis and activation of low Km phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pCl phi S-cAMP, glucagon lowered 8-pCl phi S-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pCl phi S-cAMP treatment. The results were consistent with a cAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pCl phi S-cAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Regulation of phosphoenolpyruvate carboxykinase gene transcription in H4IIE hepatoma cells: evidence for a primary role of the catalytic subunit of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

The purpose of these studies was to determine whether the catalytic subunit of cAMP-dependent protein kinase is involved in the regulation of P-enolpyruvate carboxykinase (PEPCK) gene transcription. Cyclic AMP analog pairs that preferentially stimulate either type I or type II protein kinase in a synergistic manner were used to compare regulation of mRNAPEPCK synthesis in H4IIE rat hepatoma cells with protein kinase activation in vitro. Type II protein kinase is predominant in H4IIE cells and analog pairs directed toward this isozyme resulted in a synergistic increase of mRNAPEPCK that was due to a corresponding enhancement of PEPCK gene transcription. When compared to a single analog the addition of a type II-directed analog pair reduced the total analog concentration required for maximal induction of transcription by about 30-fold. H4IIE cells have a small amount of type I kinase; pairs specific for this form of the enzyme were also effective, but to a lesser extent than those for the type II kinase. (Rp)-cAMPS, a cyclic nucleotide-dependent protein kinase antagonist, inhibited the agonist-induced increase of mRNAPEPCK in a concentration-dependent manner. The results indicate that the activation of PEPCK gene transcription by cAMP in H4IIE cells is mediated by cAMP-dependent protein kinase. Although the type II isozyme is primarily responsible, type I is also effective. These isozymes have identical catalytic subunits, hence this component presumably mediates the cAMP effect.

Animals↗
PubMed1986

Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase.

The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process.

Animals↗
PubMed1986

Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase.

By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10-20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage.

Affinity Labels↗
PubMed1985

Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and hepatocyte glycogenolysis.

Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity for the hepatocyte cAMP-dependent protein kinase isozymes and the low Km, hormone-sensitive phosphodiesterases from both cell types. No correlation was found between insulin resistance of analog-stimulated hepatocyte phosphorylase and the concentration of analog required in vitro for half-maximal activation of either type I or type II cAMP-dependent protein kinase from hepatocytes. However, a good correlation was found between insulin resistance of cAMP analog-stimulated responses and the analog I50 values for the phosphodiesterase from both cell types. Using a new method capable of measuring hydrolysis at low analog concentrations, several of those analogs which had relatively low, but not high, phosphodiesterase I50 values were shown to be directly hydrolyzed by the low Km adipocyte phosphodiesterase. The insulin inhibition of cell responses when stimulated by hydrolyzable analogs, but not by poorly hydrolyzable analogs, is best explained by insulin stimulation of the low Km phosphodiesterases from both cell types.

Adipose Tissue↗
PubMed1985

Regulatory subunit of cAMP-dependent protein kinase inhibits phosphoprotein phosphatase.

The activity of a purified high molecular weight phosphoprotein phosphatase was inhibited by purified type II cAMP-dependent protein kinase. This effect required cAMP and was obtained in the absence of ATP. The isolated type II regulatory subunits (R-subunits) from several species also inhibited the phosphatase activity in both crude extracts and purified preparations. Half maximal inhibition was observed at 0.06-0.25 microM, well within the physiological range of R-subunit concentrations. The inhibitory potency of R-subunit was greater using the thiophosphorylated form. Limited trypsinization of the R-subunit abolished the inhibitory activity. The C-subunit released the bound cAMP when combined with R-subunit, but the phosphatase did not, implying that the inhibited species is a R.cAMP-phosphatase complex. The results suggest that the R-subunit might have at least one physiological role in addition to inhibition of the C-subunit, i.e., inhibition of phosphatase. The latter would occur only when cAMP is elevated.

Adenosine Triphosphate↗
PubMed1985

cAMP-dependent protein kinase activation lowers hepatocyte cAMP.

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination. Principles for optimizing the isozyme specificity of analog combinations.

104 cAMP analogs, most of them modified in the adenine moiety, were tested as activators of cAMP-dependent protein kinase I (from rabbit or rat skeletal muscle) and kinase II (from bovine heart or rat skeletal muscle). When tested singly, only 2-phenyl-1,N6-etheno-cAMP showed a considerably (sevenfold) higher potency as an activator of kinase II than of kinase I. Analogs containing an 8-amino modification preferentially activated kinase I, some being more than 10-fold more potent as activators of kinase I than kinase II. When two analogs were combined, the concentration of one (complementary) analog required to half-maximally activate each isozyme was determined in the presence of a fixed concentration of another (priming) analog. Analogs tested in combination had been analyzed for their affinity for the intrasubunit binding sites (A, B) of isozyme I and II. The degree to which complementary analogs preferentially activated one isozyme was plotted against the mean site selectivity, i.e. (affinity A/B isozyme I X affinity A/B isozyme II) 1/2. This plot produced a straight line, the slope of which reflected the ability of the priming analog to discriminate homologous sites on the isozymes. This means that the isozyme discriminating power of an analog pair can be quantitatively predicted from the affinity of the analogs for site A and B of the two enzymes. It also means that a systematic analysis of those features of analogs imparting a high mean site selectivity or the ability to discriminate between homologous isozyme sites will facilitate the synthesis of new even more isozyme-selective analogs.

Animals↗
PubMed1985

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit.

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.

Animals↗
PubMed1985

Phosphorylation of sites 3 and 4 in rabbit skeletal muscle glycogen synthase by cAMP-dependent protein kinase.

Rabbit skeletal muscle glycogen synthase (synthase a) can be phosphorylated by 0.2 to 2.5 microM catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.5 to 3 mol of 32PO4/subunit (90,000 X g). When a complete tryptic digest of this 32P-synthase was chromatographed on reverse phase high performance liquid chromatography, it was observed that, in addition to sites 1a, 1b, and 2, site 3 was phosphorylated. The peptide containing site 5 also contained 32PO4, but sequence analysis identified a new phosphorylation site, site 4 (Arg-His-Ser-Ser(PO4)-) which precedes site 5. Phosphorylation of sites 3 and 4 became significant when the total phosphorylation stoichiometry exceeded 1.5 mol/subunit. The heat-stable protein kinase inhibitor protein or the regulatory subunit of cAMP-dependent protein kinase decreased the phosphorylation of all sites, including sites 3 and 4. Phosphorylation of all sites by the holoenzyme form of cAMP-dependent kinase was highly dependent on the presence of cAMP. These results establish that phosphorylation of these sites is due to the cAMP-dependent protein kinase itself and not to a contaminating kinase(s). Synthase b from control rabbit muscle, containing 2 to 2.5 mol of phosphate/subunit, incorporated up to 2.0 mol of 32PO4 catalyzed in vitro by the cAMP-dependent protein kinase. Again, significant 32PO4 was detected in sites 3 and 4 as well as in 1a, 1b, and 2. These results suggest that the in vivo phosphorylation of sites 1a, 1b, 2, and 3 observed after injection of epinephrine in rabbits could be due solely to the known activation of the cAMP-dependent protein kinase.

Animals↗