Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 sequences of two phosphopeptides isolated from the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase (type II) and from two of its cyanogen bromide fragments, have been determined. One phosphorylation site is a threonyl residue located approximately 180 residues from the blocked NH2 terminus. Its sequence is: -Gly-Arg-Thr-Trp-Thr(P)-Leu-Cys- and includes one of the three sulfhydryl groups present in the molecule. The second phosphorylated site within the sequence: -Val-Ser(P)-Ile-Asn- is located towards the carboxyl end of the protein where the other 2 cysteinyl residues also reside. The finding that phosphorylation of the catalytic subunit occurs on two discrete sites rather than at random suggests that it might be of physiological importance, e.g. in the regulation of enzyme activity.
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PMID:Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase. 22 92

cAMP regulates the maturation of many biochemical processes that occur during normal lung development, including the changing levels of surfactant proteins and phospholipids. We examined the effect of cAMP on the beta-adrenergic receptor concentration in the developing human lung. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, increased both the tissue cAMP content and beta-adrenergic receptor concentration in treated explants above those in untreated explants. 8-Bromo-cAMP treatment also elevated the beta-adrenergic receptor concentration of lung explants compared to that in untreated controls. These data indicate the ability of elevated cAMP to increase the beta-adrenergic receptor concentration. Both lung cAMP and beta-adrenergic receptor concentrations increase spontaneously in culture. To test for a possible causal relationship, we cultured explants with protein kinase inhibitors. We found that H-8, a preferential inhibitor of the cAMP-dependent protein kinase [protein kinase-A (PKA)], but not H-7, which inhibits PKA and protein kinase-C with similar potency, blocked the spontaneous rise in beta-adrenergic receptor concentration in human fetal lung explants, indicating that PKA activity is required for this rise in beta-adrenergic receptor concentration. Type II cells isolated from cultured lung treated with H-8 had fewer beta-adrenergic receptors than cells isolated from untreated explants. These studies show that cAMP increases the beta-adrenergic receptor concentration in human fetal lung and specifically in type II cells through a PKA-dependent mechanism, consistent with a role for cAMP in beta-adrenergic receptor regulation during normal lung development.
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PMID:Cyclic adenosine 3',5'-monophosphate increases beta-adrenergic receptor concentration in cultured human fetal lung explants and type II cells. 137 64

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
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PMID:Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa. 216 Aug 33

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).
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PMID:Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical. 254

Heterobifunctional cross-linking reagents have been introduced into the catalytic subunit of cAMP-dependent protein kinase as potential probes for identifying specific points of contact between the catalytic (C)-subunit and the type II regulatory (RII) subunit in the holoenzyme complex. Since at least one of the 2 cysteine residues in the C-subunit is known to be in close proximity to the interaction site between the C-subunit and the RII-subunit, these cysteines were chosen initially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophenacyl bromide and N-4-(azidophenylthio)phthalimide. Treatment of the C-subunit with each reagent led to the stoichiometric modification of Cys-199 and Cys-343. In each case, the modified C-subunit was still capable of forming a stable complex with the RII-subunit. Both modified C-subunits also could be covalently cross-linked to the RII-subunit; however, the mechanisms for cross-linking differed. Catalytic subunit modified by p-azidophenacyl bromide was cross-linked to the RII-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was formed and cAMP was absent. In contrast, the C-subunit modified by N-4-(azidophenylthio)phthalimide was cross-linked to the RII-subunit by a mechanism that was independent of photolysis. In this case, cross-linking was enhanced by the presence of cAMP. This cross-linking was the result of a disulfide interchange between a modified cysteine in the C-subunit and an unmodified cysteine in the RII-subunit.
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PMID:Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Heterobifunctional cross-linking reagents lead to photodependent and photoindependent cross-linking. 283 97

The intermediate filament protein vimentin was phosphorylated with cAMP-dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine-specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N-terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32P-labeled species representing amino acid position 14-72, and a minor 32P-labeled peptide representing amino acid positions 1-13. These results demonstrate that phosphorylation of sites within the N-terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.
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PMID:Cyclic AMP-dependent protein kinase-induced vimentin filament disassembly involves modification of the N-terminal domain of intermediate filament subunits. 283 68

Glycogen synthase was purified to near homogeneity from rat skeletal muscle, and was found to resemble the rabbit skeletal muscle enzyme in several respects. An apparent molecular weight (Mapp) of 86,000 was estimated from the electrophoretic mobility of the subunit on polyacrylamide gels in the presence of sodium dodecyl sulfate. Limited proteolysis of the rat synthase with trypsin resulted in the formation of species with MappS equal to 75,000, 69,000, and 67,000. The enzyme could be phosphorylated by cAMP-dependent protein kinase, phosphorylase kinase, and the cAMP-independent protein kinases, PC0.7 and FA/GSK-3. Essentially all of the phosphorylation observed occurred on serines located in two cyanogen bromide fragments, denoted CB-1 (Mapp = 13,000) and CB-2 (Mapp = 22,000). FA/GSK-3 and cAMP-dependent protein kinase phosphorylated sites in both fragments. Phosphate introduced by phosphorylase kinase was located exclusively in CB-1, and that incorporated with PC0.7 was found in CB-2. Phosphorylation by FA/GSK-3 reduced the electrophoretic mobility of the subunit, introduced heterogeneity into CB-2, and was synergistic with phosphorylation by PC0.7. To separate phosphorylation sites more completely, samples of glycogen synthase were subjected to extensive proteolysis using trypsin, followed by reverse-phase liquid chromatography. When phosphorylated by the same kinases, the pattern of fragments obtained with rat and rabbit skeletal muscle glycogen synthase were almost identical. The results presented provide strong evidence that the subunit of rat skeletal muscle glycogen synthase has at least five phosphorylation sites that are very similar, if not identical, to sites present on the rabbit muscle enzyme.
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PMID:Rat skeletal muscle glycogen synthase: phosphorylation of the purified enzyme by cAMP-dependent and -independent protein kinases. 298 12

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.
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PMID:N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases. 298 42

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.
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PMID:Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase. 301 Nov 34

The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo-cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP-dependent protein kinase is reviewed.
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PMID:Dibutyryl cyclic adenosine monophosphate reduces expression of c-myc during HL-60 differentiation. 301 82


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