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)

Of three casein phosphatases isolated from the cytosol of human cord blood erythrocytes two were cobalt-dependent, E2 and E3. In the presence of CoCl2, E2 activity was the most prominent. In addition to casein, E2 dephosphorylated phosvitin and p-nitrophenyl phosphate (p-NPP) with pH optima at 6.8-7.2 for proteins and 9.0 for p-NPP. The native enzyme had a molecular weight of 104,000 daltons after AcA-44 Ultrogel filtration. According to SDS/PAGE it consisted of two subunits, 78,000 and 15,000 daltons. The 104,000-dalton form exhibited Michaelis-Menten kinetics and had the greatest affinity for casein between protein substrates tested. Ethanol denaturated the enzyme by 80%. Optimal activation of E2 phosphatase was achieved with 5 mmol/l CoCl2 which did not affect the catalytic properties of the enzyme but did affect the rate of 'E-S' complex formation. Inorganic pyrophosphate was not inhibitory for the 104,000-dalton enzyme. Judging by all these properties the natural substrate for E2 casein phosphatase could be P-pyruvate kinase.
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PMID:Cobalt-dependent protein phosphatases from human cord blood erythrocytes. II. Further characterization of E2 casein phosphatase. 283 85

Recently, the natural vesicant cantharidin was shown to bind exclusively to and inhibit protein phosphatase 2A (PP2A) in mouse tissue extracts (Li and Casida (1992) Proc. Natl. Acad. Sci. USA 89, 11867-11870). To explore the generality of this effect in vesicant action, we measured the protein serine/threonine phosphatase activity in mouse liver cytosol (in the form of the okadaic acid inhibitable increment of p-nitrophenyl phosphate (p-NPP) phosphatase activity) in the presence of aqueous sulfur mustard or its hydrolysis product, bis(hydroxyethyl)sulfide (TDG). Sulfur mustard inhibited p-NPP hydrolysis. However, inhibition correlated with the time elapsed between thawing and the addition of mustard to the enzyme preparation, not with concentration. TDG exhibited a direct, concentration-related inhibition of p-NPP hydrolysis between 30 and 300 microM. We conclude that sulfur mustard also has an inhibitory effect on protein serine/threonine phosphatases. However, the inhibition is an effect of its non-alkykating hydrolysis product TDG, not of sulfur mustard itself.
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PMID:Possible protein phosphatase inhibition by bis(hydroxyethyl)sulfide, a hydrolysis product of mustard gas. 760 98

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate (p-NPP) and the bioluminescence assay using luciferin phosphate (L-P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC50 values of 0.9 and 6 nM using L-P and p-NPP respectively. CDP-star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC50 for the colorimetric method (IC50=2 nM [p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations < 1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels < or = 1 microg/100 g of mussel tissue which is well below the limit of 20 microg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to78% (p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC-MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC-MS all scored false negative results compared to those for the mouse bioassay in the range 20-40 microg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay > ELISA > PP-2A > LC-MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 microg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.
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PMID:Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels. 1034 Aug 30

Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.
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PMID:Biochemical characterization and inhibitory effects of dinophysistoxin-1, okadaic acid and microcystine 1-r on protein phosphatase 2a purified from the mussel Mytilus chilensis. 1569 79

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.
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PMID:Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta. 1603 40

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.
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PMID:Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina. 1712 1

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC(50) values for 21MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC(50) values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC(50) values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC(50) values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC(50) values were determined under the same experimental conditions, ensuring the quality of the data. The IC(50) values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.
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PMID:The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine. 1950 Nov 14