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 complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.
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PMID:The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle. 351 70

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.
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PMID:Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa. 352 94

Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.
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PMID:Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose. 608 42

In liver and muscle the major active phosphorylase and synthase phosphatase activity is associated with the glycogen particle. When we examined the effect of the inhibitor-1 and modulator protein on the enzyme present in crude glycogen fractions from dog liver, the phosphorylase phosphatase was not or only slightly affected. Since the enzyme isolated from the glycogen complex by DEAE-cellulose chromatography could be inhibited by inhibitor-1 as well as the modulator protein, it was assumed that an unknown mechanism or factor present in the glycogen fraction was responsible for this reduced sensitivity of the protein phosphatase. This led to the discovery (7) of the deinhibitor protein which has now been extensively purified from dog liver. The deinhibitor protein was shown to be thermostable, ethanol- and trichloroacetic acid-resistant, but non-dialyzable and it was destroyed by pronase or trypsin. The apparent molecular weight was estimated at about 17,500 in gel filtration, 8,300 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and 5,500 in sucrose density gradient centrifugation, behavior which is consistent with the assumption that the deinhibitor protein may have little ordered structure. Glycogen synthesis requires both phosphorylase and glycogen synthase as dephosphorylated enzymes. The interaction of the deinhibitor protein with the protein phosphatase brings about several effects which, when considered together, could all facilitate the dephosphorylation of glycogen synthase and phosphorylase. The protein phosphatase present in a resuspended glycogen pellet dephosphorylates inhibitor-1 in the absence of Mn2+. This ability of the phosphatase, which is lost during purification of the enzyme, can be restored upon addition of the deinhibitor protein. Owing to the association of the deinhibitor protein with the active phosphatase the enzyme becomes insensitive to inhibition by inhibitor-1 and the modulator protein, and more resistant to the conversion into the FA-ATP,Mg-dependent form, brought about by the modulator protein. During the activation of the ATP,Mg-dependent phosphatase under conditions where kinase FA is rate limiting, the deinhibitor protein increases the level without affecting the rate of activation.
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PMID:Regulation of protein phosphatase activity by the deinhibitor protein. 608 15

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

In intact red cells a CaMg-ATPase activity commensurable with that of the Ca-pump exists consisting mainly of protein kinase-protein phosphatase enzymes. The Ca:ATP stoichiometry of the Ca-pump is most probably 2:1, the deviation from this value at low [Ca] in inside-out-vesicles is possibly an artifact. Ca-affinity of the Ca-pump is low in intact red cells, where both calmodulin and calmodulin binding protein are present, and the cAMP-dependent activatory mechanism found in many other cells is inactive. Ca-affinity, however, can be enhanced by A23187, by Ca-EGTA buffers at the internal membrane surface (eliminating some structural divalent cations?), by enrichment in calmodulin and loss in calmodulin binding protein and by mild proteolytic effects on the inner surface of the membrane. Mild trypsin treatment of the external surface of the membrane increases the hydrolysis rate, but not the Ca-affinity of the Ca-pump and other CaMg-ATPases, increases membrane protein phosphorylation and protects against echinocytic shape transformation. All these findings reflect the interrelatedness of several membrane components influencing the rate and/or Ca-affinity of CaMg-ATPases.
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PMID:Ca-transport and CaMg-ATPase activity in human red cell preparations. 611 87

Studies were made on the dephosphorylation and activation of chicken liver acetyl-coenzyme-A carboxylase. The enzyme isolated by avidin-Sepharose affinity chromatography in the presence of protein phosphatase inhibitors contained 4.9 +/- 0.2 mol of alkali-labile phosphate/mol subunit and had a specific activity of 3.5 +/- 0.4 units/mg protein. The purified enzyme was dephosphorylated and activated concomitantly when incubated in the presence of protein phosphatase with a release of approximately 2 mol of phosphate/mol subunit. Limited tryptic digestion of the native and dephosphorylated forms of the enzyme (Mr 220 000) containing 4.9 and 2.9 mol of phosphate/mol of subunit, respectively, gave almost quantitatively similar polypeptides of Mr 215 000 containing 4.0 mol and 3.0 mol of phosphate per mol, respectively, which were indistinguishable by dodecylsulfate-polyacrylamide gel electrophoresis. A peptide of Mr approximately 5000 was lost from both enzymes. This result suggests that at least one of the two protein-phosphatase-labile phosphorylation sites is sensitive to trypsin. The native enzyme and those modified by protein phosphatase or by limited tryptic digestion exhibited a bi-phasic dependence on citrate. Activation of the native enzyme by protein phosphatase occurred at all the concentrations of citrate used. However, activation of the enzyme by limited tryptic digestion was found at concentrations greater than 5 mM citrate. The dephosphorylation by protein phosphatase caused an approximately fivefold activation in the enzyme activity when assayed at physiological concentrations of citrate (0.5-2.0 mM).
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PMID:Dephosphorylation and activation of chicken liver acetyl-coenzyme-A carboxylase. 613 6

Treatment of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of Mr 224 000 with 40% ethanol followed by gel-filtration on Sephadex G-150, dissociated the enzyme into an active component of Mr 31 000 and an inactive component of Mr 80 000. The inactive component reassociated with the active component, resulting in the formation of an enzyme form of Mr 123 000. A large excess of either component in the reassociation produced only this enzyme form. The ability of the inactive component to associate with the active component was lost by treatment of the inactive component with trypsin and heat (60 degrees C, 2 min) but not with DNAase and RNAase. Effects of the inactive component on the activities of the active component by the association were as followings. The inactive component: (1) stimulated slightly the 32P-H2B histone phosphatase activity in the presence of either NaCl or Mg(CH3COO)2 but inhibited strongly in the absence of the salts; (2) stimulated the 32P-H1 histone phosphatase activity in the presence of the salts; (3) inhibited the phosphorylase a phosphatase activity in the presence and absence of the salts; (4) enhanced the response to the stimulatory effects of the salts on the dephosphorylation of 32P-histone; and (5) protected the phosphorylase a phosphatase activity from inhibition by the salts.
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PMID:Isolation of an inactive component from pig heart phosphoprotein phosphatase and its reassociation with an active component. 624 55

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.
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PMID:Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions. 625 90

Plasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P]phosphorylase a or [32P]phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality. At concentrations as low as 0.1-1.0 mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or Pi. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5 M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl-and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42,000 and 32,000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.
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PMID:Phosphoprotein phosphatase associated with rat liver plasma membrane. Properties of phosphorylase phosphatase and phosphohistone phosphatase. 627 67


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