Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase activities of thyroid plasma membranes were characterized after treatment by the nonionic detergent, Triton X-100. With endogenous substrate the protein kinase activity of intact plasma membranes appeared to be cAMP independent, whereas the solubilized plasma membranes contained a cAMP-dependent protein kinase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of intact plasma membranes demonstrated approximately 30 protein bands, of which several were substrates for endogenous protein kinase, cAMP had a slight, but reproducible, stimulatory effect on some of these. In solubilized plasma membranes cAMP significantly augmented phosphorylation of at least seven of these proteins. Solubilized plasma membranes bound significantly more cAMP per mg protein than intact plasma membranes. The inability to unequivocally detect cAMP-dependent protein kinase in intact membranes using endogenous substrate probably reflects the much greater activity of the cAMP-independent enzyme activity. The protein kinase activity of intact plasma membranes which was stimulated by cAMP when histone was the substrate was primarily recovered in the solubilized plasma membranes. Most of the protein kinase activity of the intact plasma membranes was insoluble and was not augmented by cAMP. The solubilized protein kinase demonstrated the same Km values for ATP, cAMP, and MgCl2 as did the cytosolic protein kinase of the thyroid. Cytosolic and solubilized protein kinase activities were more sensitive to cAMP and cGMP stimulation when histone and protamine were used as substrates. Both enzyme activities were depressed by protein kinase modulator when histone, but not protamine and casein, were used as substrates. The protein kinase activity of insoluble plasma membranes was not inhibited by the protein kinase modulator.
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PMID:Thyroid plasma membrane-associated protein kinases: properties and substrates of solubilized and insoluble enzymes. 21 87

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.
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PMID:Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis. 21 41

The results of a series of experiments are interpreted to indicate that protein synthesis in reticulocyte lysates is not affected by the reticulocyte cAMP-dependent protein kinase. The catalytic subunit of this enzyme was isolated to apparent homogeneity. Also, the protein inhibitor of this protein kinase was isolated from muscle. Neither physiological concentrations of cAMP nor any of these protein components had a detectable effect on protein synthesis in reticulocyte lysates in the presence or absence of exogenous heme. Phosphorylation of the smallest subunit of eukaryotic initiation factor 2 or the 90,000 to 100,000-dalton peptide associated with eukaryotic initiation factor 2 kinase activity were not affected by the activity of the cAMP-dependent protein kinase under conditions in which exogenous heme has a pronounced effect on these reactions.
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PMID:No effect of cAMP on protein synthesis in reticulocyte lysates. 21 65

The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.
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PMID:Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla. 21 25

Several methods were compared for estimating the amount of regulatory subunit of an 800-fold purified Type II cAMP-dependent protein kinase from bovine heart. These methods included a reversable binding assay using either cAMP, or 8-N3-[32P]cAMP, photoaffinity labeling with 8-N3-[32P]cAMP, and autophosphorylation of the regulatory subunit of the enzyme. Although the regulatory subunit had a slightly lower affinity for 8-N3-cAMP than for cAMP, the total amount of regulatory subunit could be determined by each of the procedures examined. The results indicate that the photoaffinity analog 8-N3-[32P]cAMP is able to label quantitatively all cAMP-binding sites of the regulatory subunit of this cAMP-dependent protein kinase.
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PMID:Quantitative labeling of the regulatory subunit of type II cAMP-dependent protein kinase from bovine heart by a photoaffinity analog. 21 45

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.
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PMID:Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts. 21 99

cAMP-dependent protein kinase activity was present in a soluble TSH receptor fraction. The Km of this enzyme was 2.2 X 10(-6) M for casein substrate in the absence or presence of 10(-5) M cAMP. A [3H]cAMP-binding protein was also found in this fraction. The Ka for [3H]cAMP-binding was 0.11 X 10(6) M-1, with a total binding capacity of 3 nmol/mg protein. After fractionation using a continuous sucrose density gradient, one of the several [125I]iodobovine TSH-binding peaks corresponded to a [3H]cAMP-binding peak. After fractionation on a sucrose density gradient containing 0.4 M NaCl at pH 6.5, a major peak of protein kinase activity was shown. This protein kinase activity was stimulated by adding 10(-5) M cAMP. A peak of [3H]cAMP-binding activity corresponded to the same peak. Protein kinase activity in the receptor fraction was stimulated by adding 6 mg/ml bovine TSH. The soluble TSH receptor fraction also has an adenylate cyclase activity stimulated by TSH. These results suggest that some TSH receptors released from thyroid plasma membranes have associated adenylate cyclase activity and cAMP-dependent protein kinase activity. The receptor, cyclase, and kinase activities may exist in a functional primary receptor unit which is spontaneously released from plasma membranes.
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PMID:Adenosine 3',5'-monophosphate-dependent protein kinase activity in soluble thyrotropin receptor complex. 22 Nov 90

LH-induced desensitization of the adenylyl cyclase system in a cell-free membrane preparation from preovulatory porcine follicles exhibits a critical dependence upon Mg and ATP (1). The membrane-rich preparation was found to contain endogenous cAMP-dependent and cAMP-independent protein kinases as well as phosphorprotein phosphatases. Endogenous phosphatase activity was enchanced by by Mn2+ and dithiothreitol. The addition of either Mn2+ or dithiothreitol to the porcine follicular membrane preparation incubated under desensitizing conditions promoted a specific concentration-dependent reversal of the LH-induced desensitization of the adenylyl cyclase system. The addition of exogenous phosphoprotein phosphatase, partially purified from procine follicular cytosol, also reversed LH-induced desensitization in a concentration-dependent manner. Boiling of the phophatase preparation prevented reversal of desensitization. The addition of either exogenous beef heart cAMP-dependent protein kinase or heat-stable protein kinase inhibitor did not modify LH-induced desensitization of the follicular adenylyl cyclase system. These results provide indirect evidence that while LH-induced desensitization is not mediated by a cAMP-dependent protein kinase, reversal of desensitization can be promoted by activation of endogenous phosphatase and the addition of a homologous phosphatase preparation.
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PMID:Resensitization of the desensitized follicular adenylyl cyclase system to luteinizing hormone. 22 Nov 92

A heat-stable protein which inhibits cAMP dependent protein kinase was prepared from the bovine thyroid 100,000 X g supernatant. This protein inhibited the cAMP-dependent protein kinase both from bovine thyroid cytosol and bovine thyroid plasma membranes. The inhibitory effect was noncompetitive with histone as substrate of the cytosolic enzyme. The stimulatory modulator for cGMP-dependent protein kinase was separated from the 100,000 X g supernatant by Sephadex G-100 gel filtration. The stimulatory modulator had no stimulating effect on cAMP-dependent phosphorylation, and the inhibitory modulator of cAMP-dependent enzyme had no effect on cGMP-requiring phosphorylations. The inhibitory modulator may regulate cAMP-dependent protein kinase activity in cytosol and plasma membrane, and the stimulatory modulator for cGMP-dependent protein kinase may have a role in thyroid function independent of the cAMP-requiring system.
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PMID:Cyclic nucleotide-dependent protein kinase modulators in thyroid tissue. 22 Dec

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.
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PMID:An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization. 22 79


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