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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.
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PMID:An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. 18 23

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.
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PMID:Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo. 22 73

A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis. The enzyme has a high isoelectric point of ca. 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500. It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971), J. Biol. Chem. 246, 1986). In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors. Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+. The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP. AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction. As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F. (1960), J. Biol. Chem. 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts. Phosphorylation of beta a-casein with [gamma-32-P]ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C. (1971), eur J. Biochem. 20, 264). By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.
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PMID:Purification and properties of a yeast protein kinase. 23 75

The activity of a bifunctional enzyme, liver 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (F-2,6-Pase), which regulates the level of liver fructose-2,6-bisphosphate (F-2,6-P2), the most potent activator of PFK, is modulated by its phosphorylation rate mainly catalyzed by cAMP-dependent protein kinase A (PKA). To elucidate the action mechanism of sulfonylurea on liver F-2,6-P2 production, effects of tolbutamide on PKA-dependent phosphorylation of purified liver PFK-2/F-2,6-Phase protein and on kinase and phosphatase activities of the purified enzyme were examined in vitro. The purified enzyme was phosphorylated in the presence of the catalytic subunit of PKA, and tolbutamide inhibited the enzyme phosphorylation catalyzed by PKA in a dose-dependent manner. By adding the same dosages of tolbutamide used in the phosphorylation experiment, reduced activity of PFK-2 and increased activity of F-2,6-Pase in the presence of PKA were restored to the levels observed in the absence of PKA. On the other hand, carboxytolbutamide, an inactive metabolite of tolbutamide, had little effect on enzyme phosphorylation and activity. Our results indicate that tolbutamide inhibits a phosphorylation of the liver PFK-2/F-2,6-Pase catalyzed by PKA along with an activation of PFK-2 and an inactivation of F-2,6-Pase, leading to liver F-2,6-P2 production.
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PMID:Tolbutamide inhibits cAMP-dependent phosphorylation of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 131 65

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.
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PMID:Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle. 132 72

Phosphofructokinase was purified from muscle tissue of two different molluscs, edible snails, Helix pomatia (gastropoda), and mussels, Mytilus edulis (bivalvia). Under denaturing conditions, both enzymes had a molecular mass of 82 kDa. In the presence of ATP-Mg2+, the enzymes were rapidly phosphorylated in vitro by the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase purified from snail muscle and also by the C subunit of protein kinase from bovine heart. The extent of phosphorylation was 0.6 and 0.5 phosphate residues per subunit for the snail and the mussel phosphofructokinase, respectively. Phosphorylation of both phosphofructokinases effected a decrease in ATP inhibition at neutral or slightly acidic pH values and increased the affinity for fructose 6-phosphate. The resulting activation in the presence of suboptimum fructose 6-phosphate concentrations was more distinct for the snail enzyme. In addition, phosphorylated phosphofructokinase from mussels exhibited a marked increase in Vmax when activated by either 5'-AMP or fructose 2,6-bisphosphate.
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PMID:Phosphofructokinase from mollusc muscle is activated by phosphorylation. 165 10

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.
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PMID:Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase. 182 8

Kinetic data have been collected suggesting that heterotropic activation by fructose 2,6-bisphosphate and AMP is a result not only of the relief of allosteric inhibition by ATP but is also the result of an increase in the affinity of phosphofructokinase for fructose 6-phosphate. Modification of the Ascaris suum phosphofructokinase at the ATP inhibitory site produces a form of the enzyme that no longer has hysteretic time courses or homotropic positive (fructose 6-phosphate) cooperativity or substrate inhibition (ATP) (Rao, G.S. J., Wariso, B.A., Cook, P.F., Hofer, H.W., and Harris, B.G. (1987a) J. Biol. Chem. 262, 14068-14073). This form of phosphofructokinase is Michaelis-Menten in its kinetic behavior but is still activated by fructose 2,6-bisphosphate and AMP and by phosphorylation using the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK). Fructose 2,6-bisphosphate activates by decreasing KF-6-P by about 15-fold and has an activation constant of 92 nM, while AMP decreases KF-6-P about 6-fold and has an activation constant of 93 microM. Double activation experiments suggest that fructose 2,6-bisphosphate and AMP are synergistic in their activation. The desensitized form of the enzyme is phosphorylated by cAPK and has an increased affinity for fructose 6-phosphate in the absence of MgATP. The increased affinity results in a change in the order of addition of reactants from that with MgATP adding first for the nonphosphorylated enzyme to addition of fructose 6-phosphate first for the phosphorylated enzyme. The phosphorylated form of the enzyme is also still activated by fructose 2,6-bisphosphate and AMP.
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PMID:Fructose 2,6-bisphosphate and AMP increase the affinity of the Ascaris suum phosphofructokinase for fructose 6-phosphate in a process separate from the relief of ATP inhibition. 182 38

We have reported previously that phosphofructokinase from the liver fluke Fasciola hepatica is activated by phosphorylation with cAMP-dependent protein kinase, and that this event appears to be important in vivo for regulation of PFK (E. S. Kamemoto, L. Lan, and T. E. Mansour (1989) Arch. Biochem. Biophys. 271, 553-559). Here, we report the amino acid sequence of the single tryptic phosphopeptide generated after phosphorylation of the purified enzyme with cAMP-dependent protein kinase and [gamma 32P]ATP. Through a combination of Edman microsequence analysis, fast atom bombardment mass spectroscopy, and phosphoamino acid analysis, the sequence of the phosphorylated peptide was determined to be: R-S-T(P)-M-M-I-P-G-M-E-G-K. This sequence is not homologous to any previously determined phosphofructokinase phosphopeptides. Regulatory differences between the mammalian and parasite enzymes are discussed with particular emphasis on regulation by protein phosphorylation.
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PMID:Phosphofructokinase from Fasciola hepatica: sequence of the cAMP-dependent protein kinase phosphorylation site. 183 41

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.
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PMID:The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase. 200 78


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