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Biomedical subjects

J D Corbin

Publications and source records attributed to J D Corbin.

At least 109 records · Page 6Linked to original sources
PubMed1984

Some aspects of the phosphorylation of phenylalanine 4-monooxygenase by a calcium-dependent and calmodulin-dependent protein kinase.

A calmodulin-dependent protein kinase purified from liver catalyzed the incorporation of up to 0.7 mol of phosphate per mol subunit of phenylalanine 4-monooxygenase. The phosphorylation was accompanied by a proportional increase in the hydroxylase activity. The reaction was Ca2+-dependent and was inhibited by physiological concentrations of phenylalanine. Phenylalanine 4-monooxygenase was also a substrate for the cGMP-dependent protein kinase, but in this system phenylalanine stimulated the rate of phosphorylation to a similar extent as that observed in the reaction catalyzed by cAMP-dependent protein kinase. The hydroxylase was not a substrate for phosphorylase kinase. The calmodulin-dependent reversal of the kinase reaction in the presence of MgADP, was also inhibited by phenylalanine. Since the kinetics of the reverse reaction was the same using 32P-hydroxylase phosphorylated by calmodulin-dependent and cAMP-dependent kinases, it is likely that both kinases phosphorylate the same site on the enzyme. This conclusion was further supported by peptide mapping of tryptic and peptic digests of 32P-hydroxylase, which revealed one major phosphopeptide with enzyme phosphorylated by either kinase. The Ca2+-dependent and calmodulin-dependent phosphorylation described above may mediate the increased phosphorylation of the hydroxylase [Garrison, J. C., Johnsen, D. E., and Campanile, C. P. (1984) J. Biol. Chem. 259, 3283-3292] and its increased activity [Fisher, M. J., Santana, M. A., and Pogson, C. I. (1984) Biochem. J. 219, 87-90] recently observed in hepatocytes exposed to Ca2+-elevating agents.

Animals↗
PubMed1984

Microheterogeneity of type II cAMP-dependent protein kinase in various mammalian species and tissues.

Excluding autophosphorylated species, at least six forms of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from various mammalian tissues were identified by sodium dodecyl sulfate (SDS) gel electrophoresis of purified samples and of crude preparations photoaffinity labeled with 8-azido[32P] cAMP and by gel filtration. After autophosphorylation some heart RII forms termed type IIA (bovine, porcine, equine, and dog) shifted to a more slowly migrating band on SDS gels while others termed type IIB (rat, guinea pig, rabbit, and monkey) did not detectably shift. Both subclasses of RII exhibited variation in apparent Mr on SDS gels. Bovine and porcine heart nonautophosphorylated RII had Mr 56,000 and the autophosphorylated RII had Mr 58,000, while dog and equine heart RII had Mr 54,000 and 56,000 while rabbit and guinea pig heart RII had Mr 52,000. More than one RII was found in different tissues of the same species. Rabbit skeletal muscle contained a Mr 56,000 IIB form. Bovine lung contained almost equal amounts of a IIA form apparently identical to that of bovine heart and a Mr 52,000 IIB form similar to that which predominated in bovine brain. Rat adipose tissue, brain, and monkey heart contained predominantly a Mr 51,000 IIB form. The rat liver Mr 56,000 IIB form chromatographed differently from all other RII tested by gel filtration. Several lines of evidence indicated that the various forms of RII were not derived from one another through proteolysis or other processes. Each of the type II forms rapidly incorporated 0.3-1.0 mol of 32P per mol of subunit when incubated with [gamma-32P]ATP and C subunit. Four of the forms tested were similar in the cAMP concentration dependence for activation of their corresponding holoenzymes and inhibited C subunit about equally. Each exhibited two components of [3H]cAMP dissociation, indicating two intrachain cAMP-binding sites, and the dissociation rates for the respective sites were similar.

Adipose Tissue↗
PubMed1984

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase.

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.

Adenosine Triphosphate↗
PubMed1984

Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes.

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their Ka for protein kinase and their lipophilicity rather than with either parameter alone. Some of the most potent lipolytic analogs had I50 values for the particulate low Km cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations, some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S. R., and Corbin, J. D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response.

Adipose Tissue↗
PubMed1984

Rat adipose tissue cAMP-dependent protein kinase: a unique form of type II.

The rat adipose tissue cAMP-dependent protein kinase type II holoenzyme and regulatory (R) subunit were compared with type II from bovine heart and several other species and tissues. Adipose tissue type II was similar to the bovine heart type II by several criteria (S 20,W = 7.0, site 1 and site 2 dissociation rates for [3H]cAMP, rapid autophosphorylation and lack of MgATP inhibition of [3H]cAMP binding). However, some of its physical characteristics were similar to type I. The apparent molecular weight determined by SDS gel electrophoresis of the homogeneous adipose tissue R subunit was 51000 daltons compared to 49000 for type I and 53000-58000 for other type II R subunits. The adipose tissue holoenzyme eluted from DEAE-cellulose at an intermediate position between type I and bovine heart type II. The adipose tissue and bovine heart holoenzymes differed in several properties including Stokes radius (5.2 nm vs. 6.0 nm), calculated molecular weight (157000 vs. 181000 daltons) and frictional ratio (1.47 vs. 1.60). After autophosphorylation the adipose tissue R subunit, like type IIB forms from other species and tissues, did not shift to a higher apparent molecular weight on SDS gel electrophoresis like bovine heart type IIR subunit (a type IIA form). Even though the adipose tissue enzyme was quite similar to other type II forms in the kinetics of cAMP action, the cAMP binding sites could be shown to be different from them by the use of cAMP analogs. cAMP analogs modified at the N6 position of the adenine ring, such as N6-benzoyl-cAMP, had higher apparent Ka values for protein kinase activation for the adipose tissue enzyme than for the bovine and several other heart isozymes. cAMP analogs modified at the 8 carbon of the adenine ring showed positive cooperativity of activation for the adipose tissue enzyme but not for the bovine heart holoenzyme. The adipose tissue isozyme is the first type II form described to have a distinct kinetic characteristic.

Adenosine Triphosphate↗
PubMed1983

Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase.

The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.

Affinity Labels↗
PubMed1982

Studies on the function of the two different intrachain cyclic AMP binding sites of heart protein kinase.

The regulatory subunit of bovine heart isozyme II cAMP-dependent protein kinase contains two different intrachain cAMP binding sites which differ in their rates of cyclic nucleotide dissociation and specificity of cyclic nucleotide analogue binding. The dissociation of cAMP from intrachain Site 1 is slow compared with that from Site 2. Cyclic nucleotide analogues with C-8 alterations show a marked relative preference for Site 1, whereas N6-modified analogues select Site 2. Cyclic IMP, which prefers Site 2, activates the partially purified heart protein kinase holoenzyme as efficiently as, or more efficiently than, cAMP itself. Dissociation studies indicate that the binding of subsaturating or saturating concentrations of cAMP to isolated regulatory subunit occurs at both sites, whereas cAMP in low concentrations binds mainly to Site 1 of the holoenzyme and occupies both sites as the cAMP concentration is raised. Cyclic IMP binding to Site 2 is stimulated by the simultaneous binding of 8-Br-cAMP to Site 1, indicating that at least one function of Site 1 is cooperativity. It is concluded that Site 2 occupancy, either alone or in combination with Site 1 occupancy, is responsible for protein kinase activation. The catalytic subunit of the holoenzyme may prevent cAMP binding to Site 2, but this restraint is relieved as Site 1 becomes occupied.

Animals↗
PubMed1982

Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes.

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of cAMP-dependent protein kinase I and II were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-1-directed and site-2-directed analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]cIMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-cAMP being the most effective. Type II responded best to analogs containing a sulfur atom attached to C-8, 8-SH-cAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.

Binding Sites↗
PubMed1980

Two different intrachain cAMP binding sites of cAMP-dependent protein kinases.

The regulatory subunits of both isozymes of cAMP-dependent protein kinase bind 2 mol of cAMP/mol of monomer. cAMP dissociation studies indicate similar cAMP binding behavior for each isozyme. Each has two different intrachain cAMP binding components present in approximately equal amounts and the rate of cAMP dissociation is 5- to 10-fold slower from one site (Site 1) than from the other (Site 2). Equilibrium [3H]cAMP binding is inhibited by several competing cyclic nucleotides. Following equilibrium binding using saturating [3H]cAMP in the presence of competing nucleotide, the pattern of release of [3H]cAMP, monitored in the presence of an excess of nonradioactive cAMP, suggests site-specific selectivity of some of the cyclic nucleotides. As compared with cAMP, cIMP prefers Site 2 for both regulatory subunits, whereas N6, O2-dibutyryl-cAMP shows a similar preference only with isozyme II regulatory subunit. 8-Bromo-cAMP, 8-bromo-cGMP, and 8-azido-cAMP prefer Site 1 of both proteins. The results indicate that for each isozyme the two intrachain binding sites have different analogue specificities and cAMP dissociation rates. Site 1 or Site 2 of one isozyme has a similar but not identical cyclic nucleotide specificity and cAMP dissociation rate to the corresponding site of the other isozyme.

Animals↗