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)

Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.
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PMID:Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6. 253 37

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

A phosphoprotein phosphatase (PPase: EC 3.1.3.2) was recently purified from rat epidermis. The enzyme dephosphorylates phosphoprotein, and its properties, such as pH optimum, inhibitor spectrum, and Fe2+ activation, differ from those of other soluble phosphatases. We investigated in 2-day-old rat skin the distribution of immunologically detectable PPase and intracellular localization of PPase activity. The reaction of rabbit monospecific anti-PPase IgG was identified in granular and cornified cells by the avidin-biotin complex method. For activity staining, basic principles of the Gomori lead-salt method and azo dye technique with the substrates p-nitrophenylphosphate (p-NPP) and alpha-naphthyl phosphate (NP), respectively, were modified according to the biochemical properties of PPase activity which is resistant to formalin, Na tartrate, and NaF. Activity was detectable in granular cells including keratohyalin granules and the lower strata of cornified cells. The activity was inhibited by 1 mM CuSO4 and enhanced by a mixture of 0.5 mM FeSO4 and 1 mM ascorbic acid. We consider that PPase may be involved in dephosphorylation of histidine-rich proteins in granular and cornified cells and may play a key role in intracellular catabolism associated with epidermal cell differentiation.
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PMID:Immuno- and enzyme-histochemical detection of phosphoprotein phosphatase in rat epidermis. 253 8

Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).
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PMID:Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical. 254

Alloxan diabetes induced in white rats by intraperitoneal injection of alloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished. AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities. The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.
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PMID:Changes in the activity of enzymes, participating in glycogen metabolism of alloxan diabetic rats. 255 79

The cellular localization of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 that appears to mediate certain actions of dopamine in the mammalian brain by acting as an inhibitor of protein phosphatase 1, was studied in the kidney of several species. DARPP-32 mRNA and DARPP-32-like immunoreactivity were found in the cytoplasm of cells in the thick ascending limb of the loop of Henle. The specific dopamine DA1 agonist SKF 82526 caused a dose-dependent inhibition of Na+,K+-ATPase activity, which could be blocked by SCH 23390, a specific DA1 antagonist, and by PKI-(5-24) amide, a specific inhibitor of cAMP-dependent protein kinase. The results indicate that DA1 dopamine receptors and DARPP-32, an intracellular third messenger for dopamine, are part of the signal-transduction process for dopamine acting on renal tubule cells.
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PMID:Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and dopamine DA1 agonist-sensitive Na+,K+-ATPase in renal tubule cells. 257 60

The opening and closing of chloride (Cl-) channels in the apical membrane of epithelial cells is regulated by hormones, neurotransmitters and enterotoxins (intestine) acting through a variety of intracellular messengers, including cyclic nucleotides (cAMP, cGMP), calcium (Ca) and diacylglycerol (DAG). The chloride impermeability of epithelial membranes observed in cystic fibrosis (CF) patients does not result from a defect in the Cl- conducting properties of the channel or in channel recruitment but stems either from a defect in a key regulator of the channel, presumably a phosphoprotein, or from the hyperactivation of a channel closing mechanism, presumably a protein phosphatase or a down-regulating protein kinase (i.e. protein kinase C). In vitro phosphorylation of isolated intestinal brush border membranes has revealed the existence of a 25,000 molecular weight proteolipid (p25) acting as cosubstrate for both cGMP- and cAMP-dependent protein kinases and cross-reacting with antibodies directed against the cytoplasmic tail of the band 3 anion exchanger from erythrocytes. The putative role of p25 in Cl- channel regulation and its relationship to an unidentified GTP-binding protein recently implicated in Cl- channel activation is discussed on the basis of a regulatory model indicating potential sites of the CF defect at a molecular level.
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PMID:The molecular basis of chloride channel dysregulation in cystic fibrosis. 270 19

Requirements for the activation of Cl- conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase. A Cl- conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl- conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase. There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO4(3-), and F- into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl- conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.
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PMID:Activation of chloride conductance in pig jejunal brush border vesicles. 271 42

The activation of the ATP,Mg-dependent protein phosphatase [Fc.M] has been shown to involve a transient phosphorylation of the modulator subunit (M) and consequent isomerization of the catalytic subunit (Fc) into its active conformation (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S. -D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870). The modulator subunit constitutes the inactivating force for the enzyme, but the slow intramolecular inactivation of the phosphatase can be prevented or blocked by the addition of either the phosphorylated inhibitor-1 or Mg2+ ions. Autodephosphorylation of the modulator subunit is not prevented by the phosphoinhibitor-1, suggesting that the ATP,Mg-dependent phosphatase binds the phosphomodulator subunit in a very specific manner, different from the way it binds exogenous phosphoprotein substrates. Alternatively, the autodephosphorylation of the modulator subunit is catalyzed at a separate active site on the enzyme, which is not influenced by the binding of phosphoinhibitor-1. The phosphoinhibitor-1 does not prevent the activation of the enzyme by kinase FA when added at concentrations that totally inhibit the potential phosphorylase phosphatase activity. These results, together with other already published information, suggest separate autonomic controls of the ATP,Mg-dependent phosphatase activity by inhibitor-1 and the modulator protein through the presence of specific regulatory subunits on the enzyme.
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PMID:The ATP,Mg-dependent protein phosphatase. Regulation by inhibitor-1 or modulator protein and stabilizing role of Mg2+ ions. 282 Sep 94

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.
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PMID:Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm. 283 36


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