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 adenosine 3",5"-monophosphate (cAMP)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from bovine heart is characterized. That the ATPase activity is intimately associated with the catalytic subunit of the enzyme is suggested by the following: (i) the similar dependences of ATPase and protein kinase activities on cAMP; (ii) the dissociation of ATPase activity from the holoenzyme on addition of cAMP and its co-elution with the catalytic subunit on gel filtration chromatography; (iii) the similarity of the relative effectiveness of divalent metal ions in ATPase and protein kinase catalysis; and (iv) the correspondence of kinetically determined Km(MgATP) and Ki(MgADP) values with thermodynamic dissociation constants determined by equilibrium dialysis. The hydrolysis of ATP is stimulated 10- to 20-fold by cAMP in the holoenzyme. The molar specific activity of the catalytic subunit ATPase is approximately 0.7 min-1 with Km(MgATP) = 5 muM. MgADP is a competitive inhibitor of the reaction with a Ki value of approximately muM. The order of the relative effectiveness of metal ions for both ATPase and peptide kinase activities is Mg2+ greater than Mn2+ greater than Ca2+. A possible interpretation of these observations is that the role that the metal ion plays is more directly manifested in bond-breaking than in bond-forming.
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PMID:Cyclic AMP-dependent ATPase activity of bovine heart protein kinase. 21 18

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

Studies on the gonadotrophin-responsive adenylyl cyclase (AC) system of rabbit and porcine ovarian follicles reveal that hCG or LH-induced desensitization of the AC system can be divided into two phases: an initial, LH-specific phase and a second phase which is not specific for LH. The first phase occurs within the first hour after LH-hCG-receptor interaction, is agonist specific, and is not mediated by protein synthetic events or by cAMP. In view of our previous demonstration of the critical dependence of the LH-induced desensitizing process in cell-free membrane preparations of porcine follicles upon Mg2+ and ATP, we investigated the role of a phosphorylation reaction in the first phase of the AC desensitizing process. Porcine follicular membranes rich in LH-sensitive AC activity were found to contain the molecular requirements necessary for a phosphorylation reaction: namely, cAMP-dependent and cAMP-independent protein kinases as well as phosphoprotein phosphatases. The following lines of indirect evidence indicated that reversal or resensitization of the desenzitized AC system to LH was mediated by a dephosphorylation reaction. Activators of endogenous phosphoprotein phosphatases--Mn2+ and dithiothreitol--promoted a specific resensitization of the follicular AC system to LH. Likewise, a partially purified phosphoprotein phosphatase also resensitized the desensitized, LH unresponsive AC to LH, and boiling of the phosphatase prevented its effect. LH-induced desensitization of the AC system, on the other hand, did not appear to be mediated by a cAMP-dependent protein kinase, as evidenced both by the inability of beef heart protein to promote desensitization of AC and by the inability of an inhibitor of cAMP-dependent protein kinase to prevent LH-induced densensitization. The second phase of desensitization, which occurs after the first hour following hCG-LH-receptor interaction, is characterized by a loss of responsiveness to FSH as well as to LH and can be promoted by dibutryl cAMP (in the absence of LH). These results provide new evidence on the characteristics and molecular mechanism of LH-induced densensitization of the follicular AC system. These results indicate that the level of phosphorylation of membrane-associated components may, in part, regulate the activity of the AC system during this first phase of homologous desensitization.
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PMID:LH-induced desensitization of the adenylyl cyclase system in ovarian follicles. 22 90

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 mumol/mg of protein/min, with a Km of 4.7 microM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 microM), sodium vanadate (IC50 of 150 microM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 microM ZnCl or 10 mM NaF. Poly-(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.
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PMID:Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor. 165 33

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.
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PMID:Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein. 196 66

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.
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PMID:Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein. 215 97

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three-to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP (dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF: it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A nerve growth factor-dependent protein kinase that phosphorylates microtubule-associated proteins in vitro: possible involvement of its activity in the outgrowth of neurites from PC12 cells. 216 66

The effect of phosphorylation of pre interleukin 1 alpha (IL 1 alpha) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1 alpha (residues 64 to 271) and phosphorylated this pre IL 1 alpha in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1 alpha selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1 alpha to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100 microM was required. Unphosphorylated pre IL 1 alpha did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1 alpha did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1 alpha bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1 alpha binds to the inner surface of plasma membrane in a Ca2(+)- and phospholipid-dependent manner.
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PMID:Calcium-dependent binding of phosphorylated human pre interleukin 1 alpha to phospholipids. 239 32

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.
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PMID:Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase. 242 55

Phosphorylase phosphatase isolated from rabbit skeletal muscle can be activated in several ways. Trypsin-Mn2+ treatment of the purified Mr = 70,000 complex or addition of Mn2+ alone to the isolated inactive catalytic subunit gives enzyme species that readily dephosphorylate phosphorylase a and the type 2 regulatory subunit of cAMP-dependent protein kinase as well as synthetic phosphopeptides corresponding to the phosphorylation sites of these proteins. In contrast, enzyme activated by phosphorylation of the regulatory subunit using factor FA (glycogen synthase kinase 3) and Mg2+-ATP and thought to be of physiological significance dephosphorylates the protein substrates but not the phosphopeptides. Likewise, the active catalytic subunit isolated following FA treatment could not act on the peptides unless Mn2+ ions (maximal effect at 250 microM) were added. Mg2+ and Ca2+ could not substitute for Mn2+. Such differences in substrate specificity are not seen with p-nitrophenyl phosphate which is dephosphorylated by all forms of the phosphatase. The results suggest that the primary sequence surrounding the phosphorylation site of the substrate is not all that is necessary for recognition by the FA-activated form of the enzyme. They are interpreted in terms of constraints within the enzyme that are relaxed following exposure to Mn2+ or by the additional determinants present in larger protein substrates.
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PMID:Phosphorylase phosphatase. Comparison of active forms using peptide substrates. 282 55


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