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)

Incubation of purified vaccinia virus with gamma-(32)P-adenosine triphosphate resulted in the incorporation of (32)P into hot trichloroacetic acid-insoluble material. Enzymatic activity was completely dependent on the addition of divalent cations and was stimulated by nonionic detergents and dithiothreitol. Chemical studies demonstrated that serine and threonine residues of 15,000 molecular weight viral polypeptides were phosphorylated. In contrast, the major structural proteins were not phosphorylated or were phosphorylated to a much lesser extent. Added histones and protamine, but not serum albumin, casein, or phosvitin were phosphorylated by the partially disrupted vaccinia virus preparations. The protein kinase was tightly associated with vaccinia virus particles since the specific enzymatic activity remained constant during the final steps of virus purification, the specific activities of many different preparations of virus were similar, and the enzymatic activity cosedimented with vaccinia virus during rate zonal sucrose gradient and potassium tartrate gradient equilibrium centrifugations. Controlled degradation of vaccinia virus, with nonionic detergents and dithiothreitol, indicated that both the protein kinase and the specific phosphate acceptor proteins were located in the virus core.
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PMID:Protein kinase and specific phosphate acceptor proteins associated with vaccinia virus cores. 507 34

The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.
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PMID:Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis. 613

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.
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PMID:Characterization of a messenger RNA transport protein. 619 75

The protein kinase associated with purified herpes simplex virus 1 and 2 virions partitioned with the capsid-tegument structures and was not solubilized by non-ionic detergents and low, non-inhibitory concentrations of urea. The enzyme required Mg2+ or Mn2+ and utilized ATP or GTP. The activity was enhanced by non-ionic detergents and by Na+ even in the presence of high concentrations of of Mg2+, but not by cyclic nucleotides. The enzyme associated with capsid-tegument structures phosphorylated virion polypeptides only; exogenously added substrates (acidic and basic histones, casein, phosphovitin, protamine, and bovine serum albumin) were not phosphorylated. The major phosphorylated species were virion polypeptides (VP) 1-2, 4, 11-12, 13-14, 18.7, 18.8 and 23. VP 18.7 and VP 18.8 have not been previously detected, but may be phosphorylated forms of polypeptides co-migrating with VP 19. Of the remainder, only VP 23 has been previously identified as a capsid protein; the others are constituents of the tegument or of the under surface of the virion envelope. The distribution of the phosphate bound to viral polypeptides varied depending on the Mg2+ concentration and pH. In the absence of dithiothreitol, in vitro phosphate exchange was demonstrable in VP 23 and to a lesser extent in two other polypeptides on sequential phosphorylation frist with saturating amounts off unlabeled ATP and then with [gamma-32P]ATP. Analysis of the virion polypeptides specified by herpes simplex virus 1 X herpes simplex virus 2 recombinants indicates that the genes specifying the polypeptides which serve as a substrate for the protein kinase map in the unique sequences near the left and right reinterated DNA sequences of the L component.
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PMID:Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the polypeptides phosphorylated in the virion. 625 39

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.
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PMID:Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions. 625 90

Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)12--18 and poly(dC).oligo(dG)12--18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 micrograms/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)12--18 was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0.3 M with either activated calf thymus DNA or poly(dA).oligo(dT)12--18 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgCl2, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDS--polyacrylamide gel electrophoresis (SDS--PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.
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PMID:Human cytomegalovirus-associated DNA polymerase and protein kinase activities. 627 14

A cyclic AMP-independent protein kinase has been purified from wheat germ extracts. The enzyme catalyzes the phosphorylation of casein and phosvitin but not protamine, histone, or bovine serum albumin. However, the best substrate for the kinase appears to be that of an endogenous wheat germ protein. The kinase can utilize both ATP and GTP as phosphoryl donors. A molecular weight of 36,000-38,000 had been estimated for the kinase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by glycerol density gradient centrifugation and gel filtration in the presence of 0.5 M KCl. In the presence of low salt, however, the molecular weight of the kinase appears to double. In isoelectrofocusing, the kinase exhibits a pI of about 6.5. The activity of the kinase is strongly inhibited by spermine and heparin. Spermidine is slightly stimulatory at low concentrations but inhibitory at high concentrations. High concentrations of putrescine also inhibit the kinase activity, but not to the extent observed with the other polyamines. Both spermine and spermidine appear to enhance the kinase activity at low Mg2+ concentrations. The result suggests that these polyamines could partially replace Mg2+ for kinase activity.
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PMID:Purification and characterization of a wheat germ protein kinase. 628 29

We recently described a method for the purification of a protein kinase related to pp60src from Rous sarcoma virus-induced rat tumors (Blithe, D. L., Richert, N. D., and Pastan, I. H. (1982) J. Biol. Chem. 257, 7135-7142). In this report, we describe some of the properties of the 7200-fold purified enzyme. The purified kinase phosphorylates casein, vinculin, actin, and histone H2B, but not bovine serum albumin or ovalbumin. Protein substrates are phosphorylated exclusively on tyrosine residues. Casein was used as a substrate for more detailed analysis. The phosphorylation reaction proceeds at a linear rate for at least 40 min at 22 degrees C. Maximum enzyme activity occurs at pH 6.5 to pH 6.8 and requires the presence of either Mg2+ (5 to 10 mM) or Mn2+ (1 to 5 mM). The Km for ATP is 30 microM and the Vmax 0.03 mumol/min/mg using 0.4 mg/ml of casein as a substrate. The enzyme utilizes ATP or dATP, but not GTP as a phosphate donor in the reaction. The enzyme is inhibited by adenosine and deoxyadenosine and by their corresponding mono- and diphosphates. No inhibition of enzyme activity is observed with adenine, GTP, UTP, TTP, or CTP. The enzyme is very sensitive to increased ionic strength. Addition of 0.1 M KCl, 0.1 M NaCl, 50 mM KPO4, or 50 mM NaPO4 inhibited casein phosphorylation by 90 to 95%. Analysis of the products of the phosphorylation reaction by thin layer chromatography revealed that the src kinase phosphorylates glycerol in addition to casein or the enzyme itself.
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PMID:Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors. 628 33

The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.
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PMID:Activation of a cyclic AMP-independent protein kinase by an endogenous ATP-requiring protease from lymphosarcoma cells. 630 81

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.
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PMID:Purification and characterization of a phosvitin kinase from the thyroid gland. 683 Aug 50


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