Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium. 277 41

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

A potent anticoagulant, cerastase F-4, was purified from the venom of Cerastes cerastes. The u.v. absorption spectrum revealed a relatively high tyrosine and low tryptophan content. The molar extension coefficient and E278(0.1%) were 19,400 and 0.84, respectively. The enzyme secondary structure, as studied by circular dichroism, showed 23.6% alpha-helix, 34% beta-sheets, 19% beta-turns and 32.5% random coils. When casein was used as a substrate the optimum pH was 10.0 and the Km was 1.45 g/l. Cerastase F-4 is a metallo-enzyme that contains one mole of Ca2+ and one mole of Zn2+ per mole of protein. It is not affected by phenylmethane sulfonylfluoride or soybean trypsin inhibitor, while it is completely inhibited by 0.5 mM EDTA or ethyleneglycol bis (beta-amino ethylether) N,N,N',N'-tetraacetic acid (EGTA). Ca2+, Mg2+ and Zn2+ partially activated the enzyme under different experimental conditions. Our results suggest that Ca2+ and Zn2+ may play a role in maintaining the structural and catalytic integrity of the enzyme.
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PMID:Further characterization of the anticoagulant proteinase, cerastase F-4 from Cerastes cerastes (Egyptian sand viper) venom. 311 14

A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; pp107 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED50 = 0.2 mM) and inhibited by low concentrations of heparin (0.1-5 micrograms/ml). The Km and Vmax for the reaction were 1.46 microM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.
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PMID:Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol. 320 Feb 52

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.
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PMID:Calcium-induced associations of the caseins: a thermodynamic linkage approach to precipitation and resolubilization. 341 40

Metalloproteinase from the venom of Bothrops asper (proteinase G) is a glycoprotein with 1% neutral hexose and 3.5 moles of sialic acid per mole of protein. It hydrolyses a number of protein substrates such as casein, hemoglobin, gelatin and fibrinogen, whose alpha chain is degraded preferentially. The pH optimum of hydrolysis of casein is approximately 9.5. The protease is devoid of hemorraghic, esterolytic and amidolytic activities. The proteolytic activity of the enzyme increases by about 20% in the presence of 0.2 mM Ca2+ and Mg2+. Among the other ions tested, only Cd2+ and Fe2+ markedly decreased its activity. EDTA and cysteine are also strong inhibitors. In the presence of Ca2+ and EDTA, Zn2+ ions restored 50% of the activity. The amino acid composition shows fewer acidic residues than in related proteinases from other snake venoms.
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PMID:Characterization of a metallo-proteinase from Bothrops asper (terciopelo) snake venom. 367 44

Microtubules have been isolated from immature (3-4 weeks' old) and old (11-13 years' old) bovine brains. Quantitative studies revealed that the concentration of extractable microtubule protein per gram of wet brain decreased from 0.47 mg (immature animals) to 0.34 mg (old animals). The major components of microtubule protein (tubulin and high-molecular-weight microtubule-associated proteins) do not undergo an age-correlated change. Determination of the endogenous protein kinase activity revealed that the activity associated with "immature" calf brain microtubules was six times higher than the activity present in "old" preparations. In contrast, the stimulatory effect of cyclic AMP on protein phosphorylation in microtubules from old bovine brains exceeds nine-fold the value obtained from immature animals. After addition of casein (exogenous acceptor), the basal activities increased in both preparations without altering the age-correlated difference in the specific activity. By comparing the radioactivity pattern of sodium dodecyl sulfate polyacrylamide gels after autophosphorylation of microtubule protein with [gamma-32P]ATP, 1.5 moles of phosphate per mole of high-molecular-weight microtubule-associated protein were estimated to be incorporated in preparations from immature animals and 0.9 mole of phosphate per mole of associated protein in the experiments with "old" microtubule protein. Adenosine triphosphatase activity, associated with the high-molecular-weight microtubule-associated protein 1, was determined to be 15% reduced in preparations from old animals, compared to the activity in "young" preparations. In contrast, the guanosine triphosphatase activity increased five-fold during ageing; the higher activity of this enzyme was observed both during the initial and the steady-state phases of microtubule formation.
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PMID:Age-dependent alterations of microtubule-associated enzyme activities from bovine brain (protein kinase, adenosine triphosphatase, guanosine triphosphatase). 613 97

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.
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PMID:Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex. 628 91

Full-term newborns (FTN) plasminogen was evaluated by different techniques (affinity chromatography, immunologic technique, casein method, and chromogenic substrate). The functional activity of the FTN plasminogen was about 50% of that of the adult. This suggests the possible existence of a functional anomaly of plasminogen in FTN. FTN plasminogen aminoacids were studied, and, besides small qualitative anomalies, a decrease in amino acid residues per mole of protein and a different N-terminal amino acid were detected.
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PMID:Dysfunctional plasminogen in full-term newborn. 719 44

The fraction of secretory immunoglobulin A (sIgA) from the milk of healthy mothers was purified by sequential affinity chromatography on protein A-sepharose (in the presence of 1% triton x100), adsorbent Toyopearl HW-55 (gel-filtration), DEAE cellulose (separation of IgG and IgA antibodies), affinity sorbents with immobilized ATP and casein. The protein obtained corresponded to sIgA antibodies according to all known criteria and did not contain any protein contaminations. The ability of sIgA to phosphorylate selectively serine residues of casein (not histones) in the presence of [gamma-32P]ATP was shown. Purified kinase activity was stable at acid shock (pH 2.3), strongly interacted with immobilized antibodies against H-chain of sIgA and eluted from the sorbent with the peak corresponding to sIgA antibodies. The complex of sIgA and ATP was stable enough at the conditions of gel-filtration. Affinity modification of sIgA by chemically reactive analogs of ATP resulted in preferential modification of its light chain (2-3 mole reagent per mole of dimer form). In the condition of oligomer dissociation ATP-sepharose sorbed only the light chains of sIgA. sIgA have optimal conditions for phosphorylating activity different from those of known protein kinases. We suppose that sIgA antibodies with kinase activity are a first example of sIgA antibodies with catalytic activity. For the first time the possibility of existence of natural abzymes in a healthy human was shown. These abzymes catalyze the reaction of synthesis but not substrate degradation reaction.
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PMID:[Do catalytically active antibodies exist in healthy people? (Protein kinase activity of sIgA antibodies from human milk)]. 747 55


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