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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) from bovine tracheal smooth muscle extracts was isolated and its activity determined using two [32P]phosphorylated proteins as substrates, i.e. phosphorylated histone (H-P) and a phosphorylated muscle specific substrate protein (MS-P) for the tracheal smooth muscle protein kinase. The enzyme was purified by the use of DEAE-cellulose followed by a two stage chromatography on a histone-Sepharose affinity column. Elution from the affinity column resolved the phosphoprotein phosphatase into four activity fractions. While fractions expressed phosphatase activity against both tested substrates the relative amounts of either activity varied. The ratio of activity towards H-P to activity towards MS-P changed from 11.5 to 0.12. The characterization of four phosphoprotein phosphatase fractions was based on the differences found in the following parameters: substrate specificity; sensitivity to NaF; influences of nucleotides (ATP, 5'-AMP, cyclic AMP, cyclic GMP) and the requirement of Mn2+ for maximal activity. Mg2+, Ba2+ or Ca2+ could not substitute for Mn2+.
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PMID:Phosphoprotein phosphatase in bovine tracheal smooth muscle. Multiple fractions and multiple substrates. 20 54

When myofibrils from rat hearts were dissolved in concentrated salt solutions and reprecipitated by dilution, they contained both protein kinase (partly cyclic 3':5'-AMP-dependent) and protein phosphatase activities. Troponin-I was the major protein to be phosphorylated by the endogenous myofibril-associated kinase and by added protein kinase. Approximately 1 mole of phosphate per mole of troponin-I was incorporated from radioactive ATP, but the extent of troponin-I phosphorylation could be varied experimentally. An inverse correlation was found between protein phosphorylation and the maximum Ca2+-stimulated myofibrillar Mg2+-ATPase activity, while the amout of calcium required for half-maximum activation was proportional to the extent of protein phosphorylation. The changes in Mg2+-ATPase activity produced in vitro by protein phosphorylation were reproduced in isolated perfused rat hearts treated for short periods with L-noradrenaline (10(-6)M). The changes in myofibrillar function brought about as the result of the phosphorlyation by cAMP-dependent protein kinase suggest that the contractile response is desensitized in order to cope with the rise in intracellular Ca2+ which results from the action of catecholamines on cardiac ventricular cells.
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PMID:Cardiac myofibrillar phosphorylation and adenosine triphosphatase activity. 22 75

The activity of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid biosynthesis and malonyl-CoA production, can be regulated by several mechanisms, including multisite covalent phosphorylation, both in vitro and in intact cells. Evidence has been presented by others to indicate that a 5'-AMP-activated protein kinase (AMPK) is likely the major regulatory kinase active on ACC. While insulin is known to activate ACC in several cell types, accompanied by changes in ACC phosphorylation, the mechanism underlying this activation has been obscure. In the present study, we have examined, in Fao hepatoma cells, the effects of insulin on ACC and AMPK activity, the latter measured with a synthetic peptide corresponding to one of the phosphorylation sites on ACC for AMPK. Our results show that insulin leads to inhibition of kinase activity prior to the onset of ACC activation; the peak of maximal kinase inhibition (approximately 35% at 10 min) is seen to precede the onset of ACC activation (20 min). The inhibition of kinase activity due to insulin is observed both in the absence and presence of varying stimulating concentrations of added 5'-AMP. Both kinase inhibition and ACC activation display similar insulin sensitivity (A50 0.3 nM). Preservation of this insulin-induced kinase inhibition requires the presence of protein phosphatase inhibitors in the cell lysis buffer, suggesting that AMPK itself might be regulated by insulin-stimulated changes in kinase phosphorylation. Taken together, these data are consistent with the hypothesis that the 5'-AMP-activated protein kinase is a regulated component of the insulin signal transduction pathway and may be the major target for insulin regulation of ACC.
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PMID:Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. 134 11

The activities of several hepatic enzymes are preferentially zonated to the periportal or perivenous cells of the liver acinus. Employing dual-digitonin-pulse perfusion of rat liver in the study of acetyl-CoA carboxylase (ACC), we have identified a heretofore unrecognized feature of hepatic zonation, namely an intrahepatic gradient in enzyme specific activity. ACC activity shows a relative periportal localization in normally feeding rats, even when corrected for ACC protein mass. In contrast with results previously reported by us [Evans, Quistorff & Witters (1989) Biochem. J. 259, 821-829], the total mass of both hepatic ACC isoenzymes was not found to differ between the two hepatic zones in the present study. In perfusion eluates from fed animals, periportal ACC displays enhanced citrate reactivity and two kinetic components of acetyl-CoA reactivity; the largest periportal/perivenous gradient (5-fold) is accounted for by a species with a lower Km for acetyl-CoA. The zonal gradient in ACC maximal velocity, measured in eluates from fed rats, does not persist after ACC purification, although the isolated periportal enzyme, like dephosphorylated ACC, has a lower activation constant for citrate. Total ACC protein phosphatase activity is higher in periportal eluates, but no differences in the activities of either a 5'-AMP-activated ACC kinase or the cyclic-AMP-dependent protein kinase are noted between the hepatic zones. The induction of total hepatic ACC mass and specific activity, on fasting/refeeding with a high-carbohydrate diet, abolishes the periportal/perivenous activity gradient, largely owing to a selective activation of perivenous enzyme. Nutritional induction is also accompanied by a marked alteration in ACC acetyl-CoA kinetics and abolition of the gradient in total ACC phosphatase. These studies indicate that hepatic enzyme zonation, which is often attributed to differential expression of enzyme protein, may result from zonal variations in enzyme specific activity, owing to differences in allosteric regulation and/or covalent modification.
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PMID:Hepatic zonation of acetyl-CoA carboxylase activity. 197 69

Estrogen-induced protein was purified from rat uteri and assayed for several enzymatic activities involved in the metabolism and action of cyclic nucleotides. No adenylate and guanylate cyclase (EC 4.6.1.1 and 4.6.1.2, respectively), protein kinase (EC 2.7.1.33), and cyclic nucleotide binding activities could be demonstrated in three independent preparations of the protein. However, all three preparations exhibited significant phosphoprotein phosphatase activity (EC 3.1.3.16) on phosphorylated protamine and histones F1. This activity is optimal at neutral pH, inhibited by Zn(++), and unaffected by cyclic AMP or cyclic GMP.
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PMID:Phosphoprotein phosphatase activity associated with estrogen-induced protein in rat uterus. 415 69

A protein kinase active on fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) was demonstrated in rat liver cell sap. The protein kinase activity was stimulated by cyclic AMP and coincided with the activity of cyclic AMP-dependent protein kinase type I. In addition, three different peaks of phosphoprotein phosphatase active on [32P] phosphofructose-bisphosphatase were found on chromatography of rat liver cell sap on a DEAE-cellulose column. These phosphatases needed divalent cations for full activity. 5'-AMP, a negative modulator of fructose-bisphosphatase, had no effect on the phosphorylation-de-phosphorylation reactions of the enzyme. ATP and Ca2+ did not influence the dephosphorylation reaction of fructose-bisphosphatase.
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PMID:The demonstration in rat liver cell sap of protein kinase and phosphoprotein phosphatase active on fructose-bisphosphatase. 628 6

The influence of internal Ca2+ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca2+ current (ICa). Internal free Ca2+ ([Ca2+]i) was varied between 0.01 and 10 microM by addition of Ca(2+)-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca2+]i depressed the 5-HT effect. The dose/effect curve for the Ca2+ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants Kd1 = 0.063 microM and Kd2 = 1 microM. Addition of calmodulin (CM) antagonists (50 microM trifluoperazine or 50 microM chlorpromazine), phosphodiesterase (PDE) antagonists [100 microM isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 microM okadaic acid (OA)] in the perfusion solution caused "anticalcium" action and modified the Ca2+ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca2+ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with Kd1 = 0.04 microM and Kd2 = 0.69 microM were obtained describing the activation of the retained unblocked enzyme--PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 microM), a blocker of Ca(2+)-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca(2+)-induced suppression of the cAMP-dependent upregulation of Ca2+ channels is due to involvement of two Ca(2+)-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca2+ concentration of less than or equal to 10 microM.
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PMID:Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones. 768 96

Using AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) purified from rabbit left-ventricular heart tissue, we report direct investigation of the potential for cardiac AMP deaminase activity to be regulated by kinase-mediated phosphorylation. Rabbit heart AMP deaminase served as a substrate for Ca2+/phospholipid-dependent protein kinase (protein kinase C; PKC) exclusively; no other mammalian protein kinase phosphorylated the enzyme. PKC-dependent AMP deaminase phosphorylation was rapid, linear with respect to time and the concentrations of PKC and AMP deaminase in the reaction, and inhibitable by staurosporine. Upon phosphorylation, the apparent Km of cardiac AMP deaminase decreased from 5.6 mM to 1.2 mM, without effect on the Vmax. Whether phosphorylated or not, rabbit heart AMP deaminase was inhibited by 1.0 mM GTP, which decreased the Vmax. by approximately 50% in each case. PKC-dependent phosphorylation of cardiac AMP deaminase did not alter the enzyme's allosterism toward millimolar ATP or ADP: both nucleotides at 1.0 mM concentration decreased the apparent Km to approximately 0.5 mM. Treatment of cardiac phospho-AMP deaminase with either the protein phosphatase calcineurin or alkaline phosphatase generated a dephosphorylated form which displayed molecular and kinetic properties identical with those of the originally isolated enzyme. These data raise the possibility that a phosphorylation-dephosphorylation mechanism may regulate flux through AMP deaminase in the heart under pathological conditions, such as myocardial ischaemia, characterized by PKC activation and adenylate depletion.
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PMID:Modulation of mammalian cardiac AMP deaminase by protein kinase C-mediated phosphorylation. 838 71

The substrate specificity of the cyanobacterial dual-specificity protein phosphatase, IphP, was explored using a variety of potential substrates. The enzyme displayed phosphomonoesterase activity toward a broad range of peptide, protein, and low molecular weight organophosphate compounds. It displayed little or no hydrolase activity toward phosphodiesters, phosphoramides, carboxyl esters, or sulfoesters. However, it did display measurable pyrophosphatase activity, especially toward ADP and ATP. Among the low molecular weight phosphomonoesters, the presence of an aromatic ring either as part of the leaving group alcohol or immediately adjacent thereto, as in 5'-AMP, was a strong positive determinant for hydrolysis. Among peptide and protein substrates, a rough, but imperfect, correlation between charge character and hydrolysis was noted in which proteins and phosphorylation sites of an acidic nature seemed favored. Heparin affected IphP activity in a substrate-dependent manner. Toward small organophosphates, heparin had no significant effect, but it was inhibitory toward most protein and peptide substrates. However, toward phosphoseryl casein and MAP kinase, it enhanced activity as much as 10-fold. This enhancement was attributed to the ability of heparin to bind to these substrate proteins, as well as IphP, and recruit them to the same microenvironment.
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PMID:Substrate specificity of IphP, a cyanobacterial dual-specificity protein phosphatase with MAP kinase phosphatase activity. 865 37

1. To determine whether protein kinase C (PKC)-mediated activation of ecto-5'-nucleotidase would increase interstitial adenosine concentrations in the rat heart in vivo, we made use of the microdialysis technique and a flexibly mounted probe, which was implanted in the left ventricular myocardium and perfused with Tyrode solution. 2. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM (n = 16). Perfusion of adenosine 5'-monophosphate (AMP, 100 microM) through the probe increased the dialysate adenosine concentration markedly to 9.25 +/- 0.46 microM (n = 15). alpha, beta-Methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), an inhibitor of ecto-5'-nucleotidase, abolished the AMP-induced increase in dialysate adenosine, but did not affect the baseline level of adenosine. These observations suggest that the dialysate adenosine obtained during the perfusion with AMP, but not the baseline levels of adenosine, originated from the dephosphorylation of AMP by ecto-5'-nucleotidase. Thus, the level of adenosine measured during AMP perfusion gives an index of the activity of ecto-5'-nucleotidase in the tissue. 3. Noradrenaline (10 microM) increased the adenosine concentration measured in the presence of 100 microM AMP (i.e. the activity of ecto-5'-nucleotidase) by 38.7 +/- 9.6% (n = 5, P < 0.05), an increase which was inhibited by an antagonist of the alpha 1-adrenoceptor (prazosin, 50 microM) or of PKC (chelerythrine, 10 microM). Further application of either the alpha 1-adrenoceptor agonist methoxamine (100 microM) or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (DOG, 100 microM) also increased the adenosine concentration by 35.1 +/- 10.0% (n = 6, P < 0.05) or 40.6 +/- 8.3% (n = 5, P < 0.05), respectively. 4. The presence of okadaic acid (50 microM), an inhibitor of protein phosphatase, enhanced the noradrenaline-induced increase in adenosine concentration by 112.4 +/- 35.9% (n = 4, P < 0.05), to a level significantly (P < 0.05) greater than the increase caused by noradrenaline alone (38.7 +/- 9.6%). 5. These data provide the first evidence that alpha 1-adrenoceptor stimulation and the subsequent activation of PKC can increase adenosine concentrations in interstitial spaces of ventricular muscle in vivo, through activation of endogenous ecto-5'-nucleotidase.
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PMID:Stimulation of alpha 1-adrenoceptors and protein kinase C-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo. 928 80


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