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 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

Certain forms of cardiac failure appear to be associated with a decrease in the Ca++ sensitivity of the contractile structures, possibly due to troponin I phosphorylation. Interference of cardiotonic drugs with myofibrillar Ca++ activation instead of enhancement of Ca++ influx may therefore provide a more causal therapeutic concept in the treatment of cardiac insufficiency. APP 201-533 (3-Amino-6-methyl-5-phenyl-2(1H)-pyridinone) (the structure of which is shown below) is a novel cardiotonic agent acting neither via beta adrenoceptor stimulation nor inhibition of Na+/K+ ATPase. In the 100 microM concentration range, it increases the Ca++ sensitivity and the Ca++ affinity of functionally isolated cardiac contractile structures. This coincides with an inhibitory effect on the cAMP-dependent protein kinase from rat liver. A possible relation with the regulation of troponin I phosphorylation is discussed.
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PMID:Myofibrillar Ca++ activation and heart failure--Ca++ sensitization by the cardiotonic agent APP 201-533. 281 53

A protein phosphatase which dephosphorylates phospholamban was purified from canine cardiac cytosol. Purification involved sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, Mono Q HR 10/10, and Superose 6. The enzyme was composed of three subunits with Mr = 63,000, 55,000, and 38,000, and it could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase. Phospholamban phosphatase activity was enhanced 12-, 9-, and 3-fold by the divalent cations Mg2+, Mn2+, and Ca2+, respectively. The phosphatase was inhibited by PPi, ATP, NaF, and Pi and the degree of inhibition was different with each compound. The substrate specificity of the purified phosphatase for cardiac phosphoproteins was determined using troponin I, phospholamban, and highly enriched sarcolemmal and sarcoplasmic reticulum preparations, phosphorylated by the cAMP-dependent protein kinase. The phosphatase exhibited the highest activity with phospholamban as substrate. Thus, dephosphorylation of phospholamban by this phosphatase may participate in regulation of sarcoplasmic reticulum function in cardiac muscle.
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PMID:Purification and characterization of phospholamban phosphatase from cardiac muscle. 284 19

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a Mr of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a Mr of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 mumol/mg protein/min, and Ka values of 67 nM and 1.1 microM for calmodulin and Ca2+, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca2+, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.
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PMID:Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol. 308 63

The substrate specificity of ribosomal protein S6 kinase II (S6 K II) from Xenopus eggs was evaluated using several protein substrates and a synthetic peptide corresponding to two phosphorylation sites in ribosomal protein S6. Previous studies had shown that S6 K II is unable to phosphorylate histones, casein, or phosvitin, proteins commonly used as substrates for protein kinases. In the present study S6 K II was found to phosphorylate with a significant stoichiometry rabbit skeletal muscle glycogen synthase, cardiac and skeletal muscle troponin I, and lamin C. In addition, the S6 peptide was phosphorylated by S6 K II to the same extent as observed with the catalytic subunit of cAMP-dependent protein kinase. Studies with oocytes undergoing progesterone-induced meiotic maturation and with activated or fertilized eggs revealed identical oscillations in both S6 and lamin C kinase activity. These results indicate that S6 K II does not have an absolute specificity for S6 in vitro. Therefore, since this enzyme is regulated during the cell cycle, it may phosphorylate several other proteins of interest during mitogenic stimulation.
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PMID:Substrate specificity of ribosomal protein S6 kinase II from Xenopus eggs. 324 15

The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo.
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PMID:[Phosphorylation of troponin in the heart and skeletal muscle by Ca2+-phospholipid-dependent protein kinase]. 335 65

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.
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PMID:[Troponin from the myocardium and skeletal muscles: structure and properties]. 354 61

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca(2+) sensitivity on the adenosine triphosphatase of densensitized actomyosin.
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PMID:Phosphorylation of troponin and the effects of interactions between the components of the complex. 437 5

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.
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PMID:Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins. 622 22

The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mM free magnesium, 2 mM (magnesium-adenosine triphosphate, ionic strength 0.12, 22 degrees C, the pCa50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 mM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium.
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PMID:Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. 623 79


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