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At least 289 records · Page 16Linked to original sources
PubMed1988

1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.

We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types.

2,3-Diphosphoglycerate↗
PubMed1996

Mutants with defective phosphatase activity show no phosphorylation-dependent oligomerization of CheZ. The phosphatase of bacterial chemotaxis.

CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis. The mechanism by which the activity of CheZ is regulated is not known. We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al. (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V. M., and Macnab, R. M. (1992) J. Bacteriol. 174, 793-806), for cloning the mutant cheZ genes, overexpressing and purifying their products. We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheY approximately P), and CheY approximately P dependent oligomerization of the mutant CheZ proteins. While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheY approximately P as well as wild-type CheZ. However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheY approximately P. On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheY approximately P dephosphorylation but is not necessary for the binding to CheY approximately P, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheY approximately P, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli.

Bacterial Proteins↗
PubMed1999

A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3'-phosphoadenosine 5'-phosphate phosphatase and inositol-polyphosphate 1-phosphatase.

We report the molecular cloning in Rattus norvegicus of a novel mammalian enzyme (RnPIP), which shows both 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase and inositol-polyphosphate 1-phosphatase activities. This enzyme is the first PAP phosphatase characterized at the molecular level in mammals, and it represents the first member of a novel family of dual specificity enzymes. The phosphatase activity is strictly dependent on Mg2+, and it is inhibited by Ca2+ and Li+ ions. Lithium chloride inhibits the hydrolysis of both PAP and inositol-1,4-bisphosphate at submillimolar concentration; therefore, it is possible that the inhibition of the human homologue of RnPIP by lithium ions is related to the pharmacological action of lithium. We propose that the PAP phosphatase activity of RnPIP is crucial for the function of enzymes sensitive to inhibition by PAP, such as sulfotransferase and RNA processing enzymes. Finally, an unexpected connection between PAP and inositol-1,4-bisphosphate metabolism emerges from this work.

Amino Acid Sequence↗
PubMed1977

Alkaline phosphatase and alkaline phosphatase isoenzymes in the cat.

Feline alkaline phosphatase and alkaline phosphatase isoenzymes have been studied in tissue and serum. Alkaline phosphatase from various organs was quantitated and then subjected to cellulose acetate electrophoresis. The effects of bile duct ligation, prednisolone treatment and phenobarbital treatment on serum alkaline phosphatase was measured. The diagnostic importance of feline serum alkaline phosphatase levels is discussed in light of the results of this and other studies.

Journal Article↗
PubMed2001

Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast.

Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes. It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue. The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2). To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants. Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus. Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases. We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds. Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated. We did not detect any methylesterification of Pph3p under our conditions. Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast. The function of the PPM2 gene product remains unclear.

DNA Methylation↗
PubMed1994

Suicide inactivation of human prostatic acid phosphatase and a phosphotyrosine phosphatase.

4-Difluoromethylphenyl bis(cyclohexylammonium) phosphate was synthesized in 4 steps starting from dibenzyl phosphite and shown to be a time-dependent suicide inactivator of human prostatic acid phosphatase and the SHP protein tyrosine phosphatase. The inactivation of human prostatic acid phosphatase followed pseudo-first-order kinetics with inactivation constants of Ki = 1.0 mM; ki = 0.15 min-1 (t1/2 = 4.6 min at saturation). Phenyl phosphate protected the enzyme against inactivation, indicating that inactivation occurs in the active site. The inactivation of SHP also followed pseudo-first-order kinetics, with a t1/2 = approximately 15 min in the presence of 8.2 mM inhibitor. The mechanism of inactivation likely involves the enzymatic release of difluoromethyl phenol which rapidly eliminates fluoride, generating a quinone methide. This potent electrophile then reacts with residues at the active site of the enzyme. This inhibitor and peptidic derivatives thereof have excellent potential for selective inactivation and labeling of protein tyrosine phosphatases.

Acid Phosphatase↗
PubMed1998

Induction of a germination specific, low molecular weight, acid phosphatase isozyme with specific phosphotyrosine phosphatase activity in lentil (Lens esculenta) seeds.

A germination specific isozyme of acid phosphatase (EC 3.1.3.2) hydrolysing O-phospho-L-Tyrosine, pH optima 5.5 is induced in lentil seeds. When seeds at 0 h, 24 h and 36 h of germination are electrophorezed, native PAGE on specific enzyme staining shows several constitutive isozymes of acid phosphatases. At 48 h, an isozyme is induced which gradually decreases and then disappears at 108 h of germination. The short lived, induced isozyme is present in the embryo and seed-coat but not in the plumule and the radical. Induction of this isozyme is inhibited by cycloheximide and actinomycin-D and increased by plant growth regulators such as heteroauxin and gibbrellic acid treatment during germination. The induced isozyme is a single 30 kD polypeptide, with subunit molecular mass of 25 kD, shows activity for O-phospho-L-Tyrosine. It is strongly inhibited by vanadate (microM), molybdate, tungustate as also by iodoacetate, p-chloromercuribenzoate and diethylpyrocarbonate. This study shows for the first time that the germination induced low molecular weight Acid phosphatase is a Tyrosine phosphatase super family class IV enzyme, having a role in cellular differentiation and development during seed germination.

Acid Phosphatase↗
PubMed1981

Phosphoprotein phosphatase activity of bovine intestinal alkaline phosphatase.

The phosphoprotein phosphatase activity of a commercial preparation of bovine intestinal alkaline phosphatase (EC 3.1.3.1) was examined using phosvitin and dentine phosphoprotein as substrates. Over 90% and 70% of the phosphorus from dentine phosphoprotein and phosvitin were hydrolyzed in 2 h. The optimum pH of the enzyme for the dephosphorylation of phosvitin and dentine phosphoprotein was nearly 6. No protein phosphatase activity was observed when the alkaline phosphatases from bovine liver and pulp were investigated.

Alkaline Phosphatase↗
PubMed1990

Purification and characterization of a low-molecular-weight acid phosphatase--a phosphotyrosyl-protein phosphatase from bovine heart.

A low-molecular-weight acid phosphatase that is representative of a group recently shown to be phosphotyrosyl protein phosphatases was purified to homogeneity from bovine heart. The enzyme was a monomer with a molecular mass of 18 kDa and had an isoelectric point of 7.0. The absorption coefficient, E1% 1cm was 9.65 at 280 nm. The enzyme had pH optima of 5.3 and 6.0 with the substrates p-nitrophenyl phosphate and tyrosine phosphate, respectively. When measured at pH 5 and 37 degrees C, the enzyme had specific activities of 114 and 86 mumol min-1 mg-1 for p-nitrophenyl phosphate and tyrosine O-phosphate, respectively, while the Km values were 0.38 and 14 mM. The enzyme was highly specific for aryl monophosphate esters and showed little or no activity toward aliphatic phosphate esters, with the remarkable exception of flavin mononucleotide (FMN) and certain of its structural analogs. As shown by 31P NMR data, the activity toward FMN was due to the hydrolysis of one of the eight components present in the (commercial) sample. Both molybdate and vanadate were potent inhibitors, with inhibition constants of 37 and 29 microM, respectively; tartrate and fluoride had little effect on enzymatic activity. A two-stage reversible denaturation of the enzyme by guanidine HCl was observed with midpoints of 0.25 and 1.75 M, respectively. The amino acid composition was homologous to the low-molecular-weight acid phosphatases from other tissue. The enzyme showed immunological cross-reactivity against low-molecular-weight human liver acid phosphatase. There were 7 or 8 accessible cysteines on the monomeric protein and at least one was essential for enzyme activity. The enzyme also had phosphotransferase activity, for example transferring phosphate from p-nitrophenyl phosphate to a wide variety of alcohol acceptors.

Amino Acids↗
PubMed1976

An unusual alkaline phosphatase isoenzyme in a patient with concomitant marked elevation of acid phosphatase without clinical evidence of prostatic carcinoma.

An 81-year-old male with congestive heart failure and prostatic hypertrophy was found to have markedly elevated serum acid phosphatase (EC 3.1.3.2) and moderately elevated serum alkaline phosphatase (EC 3.1.3.1). Alkaline phosphatase isoenzyme analysis was performed to determine the organ source of the enzyme. There was an unsual slow-migrating alkaline phosphatase isoenzyme band in the serum of this patient suggestive of hepatic origin by a variety of biochemical tests.

Acid Phosphatase↗
PubMed1989

Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid.

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin-dependent multiprotein kinase (CaM-MPK) and cyclic AMP-dependent protein kinase were shown to be Ser-19 and Ser-40, respectively, while Ser-40 was also phosphorylated slowly by CaM-MPK. (ii) Type 2A and type 2C phosphatases accounted for approximately 90% and approximately 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+ (1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.

Adrenal Medulla↗
PubMed1993

Inhibition of protein phosphatases activates glucose-6-phosphatase in isolated rat hepatocytes.

Incubation of hepatocytes in the presence of microcystin-LR, okadaic acid, calyculin A (inhibitors of protein phosphatases PP1 and PP2A) or microcystin-RR (a specific inhibitor of PP2A) activated glucose-6-phosphatase both in the supernatant and in intact or disrupted microsomes. Puromycin, an inhibitor of protein synthesis, totally suppressed this activating effect, suggesting the involvement of protein phosphatases in the regulation of glucose-6-phosphatase synthesis.

Animals↗
PubMed1983

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast.

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).

Kinetics↗
PubMed2006

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.

Acid Phosphatase↗
PubMed1988

Molecular cloning of rat phosphoprotein phosphatase 2A beta cDNA and increased expressions of phosphatase 2A alpha and 2A beta in rat liver tumors.

A cDNA clone coding for an isotype of the catalytic subunit of rat phosphoprotein phosphatase 2A was isolated. The deduced amino acid sequence of the clone was different at 8 positions from that of rat phosphatase 2A alpha determined in a previous study. The deduced amino acid sequence of the clone was, however, identical to that of human phosphatase 2A beta and differed only at one position from that of rabbit 2A beta. Thus, the isolated cDNA was identified as a clone coding for rat phosphatase 2A beta. Using a 2A beta specific probe, two kinds of transcripts were detected in rat liver: a major 2.0 kb mRNA transcript and a minor 1.4 kb mRNA transcript. These transcripts were both greatly increased in rat liver tumors induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) regardless of the carcinoma or hyperplastic nodule.

Amino Acid Sequence↗
PubMed1998

Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia.

A novel alkaline phosphatase, designated PiALP, has been purified and characterized from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease. The enzyme was a homodimer of apparently identical subunits of Mr 54 kDa. Thiol-reducing agents completely inhibited the purified enzyme. The enzyme was highly stable even at 80 degrees C. It exhibited substantial activity against tyrosine-phosphate-containing Raytide. The phosphatase activity was sensitive to orthovanadate and Zn2+ but highly resistant to okadaic acid. The amino acid sequence of peptides derived from PiALP showed a high degree of identity (65%) with alkaline phosphatases from Zymomonas mobilis and Synechococcus. The present results imply that PiALP might represent a new family of alkaline phosphotyrosyl phosphatases which has not been described previously.

Alkaline Phosphatase↗
PubMed2001

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

Animals↗
PubMed2003

Identification of myotubularin as the lipid phosphatase catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP.

Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in myotubularin result in the human disease X-linked myotubular myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human myotubularin were identified. Association between 3-PAP and myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with myotubularin led to attenuation of the plasma membrane phenotype, associated with myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an "adapter" for myotubularin, regulating myotubularin intracellular location and thereby altering the phenotype resulting from myotubularin overexpression.

Amino Acid Sequence↗