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)

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.
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PMID:Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver. 0 49

The regulatory mechanism of a phosphoprotein phosphatase (EC 3.1.3.16), which is considered to catalyze the dephosphorylation reaction of several phosphoproteins (glycogen synthetase-D (EC 2.4.1.11), phospho-form of phosphorylase b kinase (EC 2.7.1.38), phosphohistone and phosphorylase a (EC 2.4.1.1)), was studied with partially purified preparations from rabbit skeletal muscle. Time- and temperature-dependent inactivation and reactivation of phosphohistone phosphatase, as well as phosphorylase phosphatase (EC 3.1.3.17), were observed on pre0incubation of the enzyme(s) with ATP, and subsequent incubation with divalent metal ions (Mg2+, Mn2+, or Co2+) without any change of molecular size. Manganese, however, instantly restored the activity of the ATP-inactivated enzyme, and increased the maximal velocity of the enzyme while decreasing its affinity to phosphorylase a. However, the metal ion inhibited the reactivated enzyme competively with respect to phosphorylase a. It is suggested that phosphoprotein phosphatase(s) is a metalloenzyme, and that ATP results in a conformational change of the enzyme protein in such a way that a metal ion can be easily released due to the chelating effect of ATP, or incorporated (in the presence of excess metal ions) into the enzyme protein.
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PMID:Inactivation and reactivation of phosphoprotein phosphatase of rabbit skeletal muscle. Role of ATP and divalent metal ions. 16 88

1. Troponin I isolated from fresh cardiac muscle by affinity chromatography contains about 1.9 mol of covalently bound phosphate/mol. Similar preparations of white-skeletal-muscle troponin I contain about 0.5 mol of phosphate/mol. 2. A 3':5'-cyclic AMP-dependent protein kinase and a protein phosphatase are associated with troponin isolated from cardiac muscle. 3. Bovine cardiac 3':5'-cyclic AMP-dependent protein kinase catalyses the phosphorylation of cardiac troponin I 30 times faster than white-skeletal-muscle troponin I. 4. Troponin I is the only component of cardiac troponin phosphorylated at a significant rate by the endogenous or a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. 5. Phosphorylase kinase catalyses the phosphorylation of cardiac troponin I at similar or slightly faster rates than white-skeletal-muscle troponin I. 6. Troponin C inhibits the phosphorylation of cardiac and skeletal troponin I catalysed by phosphorylase kinase and the phosphorylation of white skeletal troponin I catalysed by 3':5'-cyclic AMP-dependent protein kinase; the phosphorylation of cardiac troponin I catalysed by the latter enzyme is not inhibited.
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PMID:The phosphorylation of troponin I from cardiac muscle. 17 90

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.
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PMID:Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle. 19 24

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.
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PMID:Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle. 19 25

1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There uas no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.
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PMID:Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle. 20 Dec 93

Properties of the ATP-dependent calcium transport system of heart sarcolemma are presented. Calcium accumulation (with oxalate) in sarcolemma was increased due to cAMP-dependent protein kinase and phosphorylase b kinase. Protein kinase increased the Vmax of the sarcolemmal calcium accumulation without any detectable effect on the affinity for Ca2+. Both kinases failed to stimulate calcium binding. Protein kinase catalyzed phosphorylation of membrane proteins of molecular weights of 100,000, 25,000, and 14,000. Phosphorylase b kinase also catalyzed phosphorylation of these proteins. Protein kinase stimulated ATPase activity of sarcolemma. Sarcolemma contained endogenous protein kinase and protein phosphatase activities.
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PMID:Characteristics of heart sarcolemmal calcium transport system and effect of protein kinase on sarcolemmal calcium accumulation. 20 83

The effects of streptozotocin-induced diabetes and of insulin supplementation to diabetic rats on glycogen-metabolizing enzymes in liver were determined. The results were compared with those from control animals. The activities of glycogenolytic enzymes, i.e. phosphorylase (both a and b), phosphorylase kinase and protein kinase (in the presence or in the absence of cyclic AMP), were significantly decreased in the diabetic animals. The enzyme activities were restored to control values by insulin therapy. Glycogen synthase (I-form) activity, similarly decreased in the diabetic animals, was also restored to control values after the administration of insulin. The increase in glycogen synthase(I-form) activity after insulin treatment was associated with a concomitant increase in phosphoprotein phosphatase activity. The increase in phosphatase activity was due to (i) a change in the activity of the enzyme itself and (ii) a decrease in a heat stable protein inhibitor of the phosphatase activity.
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PMID:The effect of streptozotocin-induced diabetes and of insulin supplementation on glycogen metabolism in rat liver. 20 91

Inhibitor-1 from rabbit skeletal muscle was phosphorylated by protein kinase dependent on adenosine 3' :5'-monophosphate (cyclic AMP), but not by phosphorylase kinase or by glycogen synthetase kinase-2. Protein phosphatase-III, isolated and stored in the presence of manganese ions to keep it stable, was in a form which catalysed a rapid dephosphorylation and inactivation of inhibitor-1. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 0.7 micron, V(rel) = 40] were comparable to those for the dephosphorylation of phosphorylase kinase [Km =1.1 micron, V (rel) = 62] and phosphorylase [Km = 5.0 micron, V (rel) = 100]. The dephosphorylation of inhibitor -1 was inhibited by inhibitor-2, indicating that it was catalysed by protein phosphatase-III, and not by another enzyme that might be contaminating the preparation. When protein phosphatase-III was diluted into buffers containing excess EDTA, it lost activity initially, but after 90 min, the activity reached a plateau that remained stable for at least 20h. The initial loss in activity varied with the substrate that was tested; it was 20-30% with phosphorylase a, 50-60% with phosphorylase kinase and greater than or equal to 95% with inhibitor-1. This form of protein phosphatase-III was inhibited by inhibitor-1 in a noncompetitive manner, and the Ki for inhibitor-1 was 1.6 +/- 0.3 nM. The phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthetase phosphatase activities of protein phosphatase-III were inhibited in an identical manner by inhibitor-1. This result emphasizes the potential importance of inhibitor-1 in the regulation of glycogen metabolism, since it can influence the state of phosphorylation of three different enzymes. The formation of the inactive complex between inhibitor-1 and protein phosphatase-III was reversed by incubation with trypsin (which destroyed inhibitor-1, but not protein phosphatase-III) or by dilution of the inactive complex. Kinetic studies, using the form of protein phosphatase-III which dephosphorylated inhibitor-1 very rapidly, demonstrated three unusual features of the system: (a) inhibitor-1 was still as powerful and inhibitor of the dephosphorylation of phosphorylase a and phosphorylase kinase a even under conditions where it was being rapidly dephosphorylated; (b) inhibitor-1 was not an inhibitor of its own dephosphorylation; (c) phosphorylase a did not effect the rate of dephosphorylation of inhibitor-1 even when it was present in a 50-fold molar excess over inhibitor-1. The result of these three properties is that inhibitor-1 is preferentially dephosphorylated by protein phosphatase-III even in the presence of a large excess of other phosphoprotein substrates. Inhibitor-1 was also dephosphorylated by protein phosphatase-II. The kinetic constants for the dephosphorylation of inhibitor-1 [Km = 2.8 micron, V (rel) = 200] and the alpha-subunit of phosphorylase kinase [Km = 3.7 micron, V (rel) = 100]were comparable...
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PMID:The regulation of glycogen metabolism. Phosphorylation of inhibitor-1 from rabbit skeletal muscle, and its interaction with protein phosphatases-III and -II. 20 45

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.
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PMID:Phosphorylase kinase from human polymorphonuclear leukocytes. 44 42


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