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)

Histidine-containing protein (HPr) of gram-positive bacteria was found to be phosphorylated at a seryl residue (P-ser-HPr) in an ATP-dependent reaction catalyzed by a protein kinase (J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. U.S.A. 80:6790-6794, 1983). Here we describe the purification and characterization of a soluble enzyme of Streptococcus faecalis which splits the phosphoryl bond in P-ser-HPr. The enzyme has a molecular weight of ca. 7.5 X 10(4), as determined by its migration behavior on a Sephacryl S-200 column. On native polyacrylamide gels the purified enzyme produced only one protein band. On sodium dodecyl sulfate-polyacrylamide gels we found one major protein band of molecular weight 2.9 X 10(4) and two minor protein bands of molecular weights 2.3 X 10(4) and 7 X 10(4). Fructose 1,6-diphosphate, which stimulated the ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, had no effect on the phosphatase activity. Other glycolytic intermediates also had no effect. However, inorganic phosphate, which inhibited the ATP-dependent HPr kinase, stimulated the P-ser-HPr phosphatase. EDTA at a concentration of 0.1 mM completely inhibited the phosphatase. Divalent cations like Mg2+, Mn2+, and Co2+ overcame the inhibition by EDTA. Fe2+, Zn2+, and Cu2+ had no effect, whereas Ca2+ slightly inhibited the phosphatase. ATP was also found to inhibit the phosphatase. Under conditions in which ATP severely inhibited the phosphatase, ADP was found to have no effect on the enzyme activity. Besides P-ser-HPr of S. faecalis, the phosphatase was also able to hydrolyze the phosphoryl bond in P-ser-HPr of Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, and Lactobacillus casei. Phosphoenolpyruvate-dependent o-nitrophenyl-beta-D-galactopyranoside phosphorylation, catalyzed by the S. aureus phosphoenolpyruvate:lactose phosphotransferase system, was about 150-fold decreased in the presence of P-ser-HPr of S. aureus, as compared with HPr. However, when P-ser-HPr was first incubated with P-ser-HPr phosphatase to allow complete hydrolysis of the phosphoryl bond, it had the same activity as HPr. Besides this cytoplasmic phosphoprotein phosphatase, we detected a membrane-bound phosphatase which also hydrolyzed the phosphoryl bond in P-ser-HPr.
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PMID:Streptococcal phosphoenolpyruvate: sugar phosphotransferase system: purification and characterization of a phosphoprotein phosphatase which hydrolyzes the phosphoryl bond in seryl-phosphorylated histidine-containing protein. 299 39

Protein phosphorylation was examined in cytosolic extracts of adult rat anterior pituitary. In the presence of both cyclic AMP and calmodulin, the phosphorylation of a Mr 22,000 protein was markedly stimulated. Cyclic AMP and calmodulin must both be present in order for this effect to be observed; cyclic GMP does not substitute for cyclic AMP, and the effect is abolished by either trifluoperazine or the heat-stable inhibitor of cyclic AMP-dependent protein kinase. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that there are three molecular species of the Mr 22,000 phosphoprotein, with pI values ranging from 6.8 to 8.1. Phosphorylation of this protein is maximally stimulated by 5 microM cyclic AMP and 5.7 microM calmodulin. The effect of cyclic AMP plus calmodulin is enhanced by preincubation and requires a divalent cation; maximal phosphorylation takes place at 100 microM Mn2+, although higher concentrations of Mg2+ and Co2+ support an equivalent degree of phosphorylation. Cyclic AMP plus calmodulin-dependent protein phosphorylation was not detected in other rat tissues surveyed, including brain, testes, adrenal, kidney, liver, spleen, skeletal muscle, pineal, or posterior pituitary. These results help to explain the previous findings of Brattin and Portanova (Brattin, W.J., Jr., and Portanova, R. (1981) Mol. Cell. Endocr. 23, 77-90) of in vivo but not in vitro phosphorylation of three Mr 20,000 anterior pituitary proteins and indicate a possible point of convergence for calcium and cyclic AMP actions in the anterior pituitary.
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PMID:Calmodulin plus cyclic AMP-dependent phosphorylation of a Mr 22,000 pituitary protein. 299 45

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.
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PMID:Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides. 300 44

Three cyclic AMP (cAMP)-dependent protein kinases, designated A1, A2, and B, were isolated from the liver fluke Fasciola hepatica using Phenyl-Sepharose and DEAE-cellulose chromatography. These enzymes differed with respect to activation by cAMP and their molecular weights. The half-maximal activation constant for cAMP-dependent protein kinases A1 and B was 20 nM, while that of A2 was about five-fold higher (110 nM). The estimated molecular weights for cAMP-dependent protein kinases A1 and A2 (both 98,000) suggest a dimeric form for these enzymes; whereas, the higher molecular weight for cAMP-dependent protein kinase B (187,000) indicates that this enzyme is a tetramer. The physical and kinetic properties of the catalytic subunit of fluke cAMP-dependent protein kinase were similar to those reported for the mammalian enzyme. The molecular weight of the catalytic subunit was estimated to be 41,000. The pH optimum for the enzyme was 6.0, 6.5, or 7.0 when casein, histone, or protamine were used as substrates. The protein substrate specificity was in the order histone greater than arginine-rich histone greater than casein greater than protamine greater than lysine-rich histone. Free Mg2+ 'stimulated' enzyme activity at low concentrations (0.5 to 5 mM), whereas at higher concentrations (greater than 5 mM) it became inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could substitute for Mg2+. Kinetic studies indicated that the reaction mechanism of this enzyme is sequential and that MgATP and MgADP are competitive ligands. Reconstitution experiments using the subunits of fluke and bovine heart cAMP-dependent protein kinase showed that there is sufficient structural homology between these enzymes such that the catalytic subunit from one species can combine with the regulatory subunit of the other species to form inactive holoenzyme. Thus, the present results indicate that cAMP-dependent protein kinase from F. hepatica is similar but not identical to the mammalian enzyme.
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PMID:Partial purification and characterization of cAMP-dependent protein kinase from Fasciola hepatica. 303 68

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.
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PMID:Properties of neurofilament protein kinase. 346 81

Primary cultures of airway smooth muscle cells were exposed to histamine, and intracellular free calcium transients were measured by the calcium-sensitive dye fura-2. Stimulation with 100 microM histamine resulted in a rise in intracellular calcium from an unstimulated level of 178 +/- 25 to 497 +/- 154 nM Ca2+ (SE; n = 14) and a return to base-line free calcium concentration within 1 min of stimulation. Pretreatment of cells with the H1 receptor blocker pyrilamine (2.5 microM) abolished the response; however, the calcium transient was not altered by pretreatment with the H2 blocker cimetidine (50 microM), by chelation of external calcium, or by pretreatment with 2 mM Co2+ or 5 microM nifedipine. Activation of protein kinase c by 200 nM phorbol 12-myristate 13-acetate (PMA) resulted in no detectable rise in cytosolic calcium but completely blocked the release of internal calcium by histamine. We conclude that 1) histamine causes a transient rise of cytosolic calcium in airway smooth muscle, 2) the rise in cytosolic calcium is mediated by H1 receptor coupling that triggers release of internal calcium stores, and 3) activation of protein kinase c blocks the histamine-induced release of intracellular calcium.
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PMID:Histamine-induced calcium release and phorbol antagonism in cultured airway smooth muscle cells. 366 94

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.
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PMID:Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes. 370 Mar 91

A tyrosine protein kinase activity has been partially purified from calf thymus using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Detergent extracts of calf thymus possessed only low levels of specific peptide phosphorylating activity when assayed at low ionic strength. The inclusion of NaCl at a concentration of 2 M stimulated endogenous tyrosine protein kinase activity, while the activity of other endogenous kinases was inhibited. This sensitivity to NaCl was retained following partial purification of the enzyme. The phosphorylation of other substrates such as casein or the R-R-SRC peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) by the tyrosine protein kinase was less sensitive to NaCl. Phosphorylation of the PK-1 peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) by the purified catalytic subunit of cAMP-dependent protein kinase was inhibited by NaCl. The effect of NaCl on angiotensin I phosphorylation could be mimicked by KCl or sodium acetate. The principal effect of NaCl was to increase the Vmax of the enzyme for the phosphorylation of angiotensin I. At low ionic strength, Mn2+ and Co2+ were the preferred required divalent cations. At elevated NaCl concentrations Mg2+ was preferred, with half-maximal activation occurring at 35 mM Mg2+. By conducting peptide phosphorylation assays in the presence of elevated levels of Mg2+ and NaCl, tyrosine protein kinase activity can readily be detected in extracts from cell lines that express low levels of the enzyme.
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PMID:Properties of a tyrosine protein kinase from calf thymus. Response to ionic strength and divalent cations. 387 56

The adenovirus endogenous protein kinase specifically phosphorylates capsid protein IIIa in the presence of Mg2+, utilizing either ATP or GTP as a phosphate donor. When Mn2+ is substituted for Mg2+, in the presence of ATP, phosphorylation of IIIa is enhanced by 2-3 fold. However, in addition to IIIa phosphorylation, the core proteins V and VII are now phosphorylated. A similar finding is made when Co2+ is used instead of Mg2+. Further, when Mn2+ or Co2+ is substituted for Mg2+, the phosphoamino acid residue profile is changed, viz., instead of only phosphoserine being labeled, phosphothreonine is now extensively labeled. These results are specific for the above endogenous viral proteins, since when the viral protein kinase is used to phosphorylate added exogenous substrates such as casein, no enhancement of phosphorylation nor any change in phosphoamino acid profile is achieved by substituting Mn2+ for Mg2+. It is thus likely that MnATP or CoATP somehow interacts differently with adenovirus structural proteins than does MgATP and this facilitates their accessibility to the enzyme.
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PMID:An unexpected effect of divalent cations on the adenovirus endogenous protein kinase: alteration in the specificity of phosphorylation. 399 34

Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction.
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PMID:Protein kinase and its endogenous substrates in coated vesicles. 614 72


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