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PubMed2002

Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.

Chromatography, High Pressure Liquid↗

PubMed2004

Differential effects of prenyl pyrophosphates on the phosphatase activity of phosphotyrosyl protein phosphatase.

Phosphotyrosyl protein phosphatase (PTPase) 1B was purified from human placenta. Immunoprecipitation analysis revealed that the isolated PTPase 1B appears as a complex with the receptor for protein kinase C (RACK1) and protein kinase C (PKC)delta. The abilities of PTPase 1B and PKCdelta to associate with RACK1 were reconfirmed by an in vitro reconstitution experiment. The E. coli expressed and biotinylated mice-RACK1-encoded fusion protein was capable of recruiting PTPase 1B and PKCdelta in the antibiotin immunoprecipitate as a complex of PTPase 1B/RACK1/PKCdelta. Thus PTPase 1B enzyme preparation was subjected to further purification by selective binding of PTPase 1B onto PEP(Taxol) affinity column in the absence of ATP. The purified PTPase 1B enzyme exihibited dose-dependent phosphatase activity towards [gamma-(32)P]-ATP labeled mice beta-tubulin-encoded fusion protein. The dephosphorylation reaction with PTPase 1B was enhanced with geranylgeranyl pyrophosphate, but not with farnesyl pyrophosphate. Interestingly, additional incubation of the purified PTPase 1B enzyme preparation with RACK1, geranylgeranyl pyrophosphate failed to modulate the dephosphorylation activity of PTPase 1B. In contrast, the enhancement effect of farnesyl pyrophosphate on the kinase activity of PKCdelta was sustained in the presence of RACK1. That is, farnesyl pyrophosphate may function as a signal to induce the kinase activity of PKCdelta in PTPase 1B/RACK1/PKCdelta complex but geranylgeranyl pyrophosphate may not for PTPase 1B. J. Exp. Zool. 301A:307-316, 2004.

Animals↗

PubMed1997

Association of protein phosphatase-1delta with the retinoblastoma protein and reversible phosphatase activation in mitotic HeLa cells and in cells released from mitosis.

The retinoblastoma gene product (pRb) is dephosphorylated at the exit from mitosis and protein phosphatase-1 (PP1) seems to be responsible for such dephosphorylation. Three isoforms of PP1 exist in mammalian cells, alpha, gamma1 and delta, with differential subcellular localization and potentially different targeting subunits and functions. In order to identify which isoform dephosphorylates pRb, we used isoform-specific antibodies and analyzed the association of the PP1 isoforms with pRb in nocodazole-blocked (mitotic) HeLa cells and in cells released from the mitotic block (early G1). PP1delta was found associated with the pRb immunoprecipitated from a mitotic cell extract, whereas neither PP1gamma1 nor PP1alpha were detected. In G1 cells progressively less pRb and of lower Mr was detected in anti-PP1delta immunocomplexes, and pRb had almost disappeared by 8 h. The PP1 associated with pRb was inactive at mitosis, but underwent a quick activation as cells exited from mitosis, with a peak at 1 h. Then the activity decreased progressively and disappeared by 8 h. [32P]labeled pRb, obtained from G2 cells, was dephosphorylated "in vitro" by PP1delta obtained from early G1 cells. Altogether, the results indicated that PP1delta associated with pRb and may be responsible for the phosphatase activity detected in the pRb complexes, supporting the hypothesis that PP1delta may be the isoform that dephosphorylates pRb.

Blotting, Western↗

PubMed2002

Phosphatase and oxygen radical-generating activities of mammalian purple acid phosphatase are functionally independent.

Bone-resorbing osteoclasts and activated macrophages express large amounts of tartrate-resistant acid phosphatase (TRAP), an iron-containing enzyme with unknown biological function. We studied acid phosphatase (AcP) and reactive oxygen species (ROS)-generating activities of recombinant rat TRAP. pH optimum was 4.5 for AcP activity and 6.5 for ROS-generating activity. Replacement of His113 and His216 by site-directed mutagenesis severely inhibited AcP activity, but had no significant effects on ROS-generating activity. Substrate specificity was not affected by the mutations. These results suggest that AcP and ROS-generating activities of TRAP are functionally independent.

Acid Phosphatase↗

PubMed1995

Structure and chromosomal localization of the human gene of the phosphotyrosyl phosphatase activator (PTPA) of protein phosphatase 2A.

The PTPA gene encodes a specific phosphotyrosyl phosphatase activator of the dimeric form of protein phosphatase 2A. PTPA, cloned from human genomic libraries, is encoded by one single-copy gene, composed of 10 exons and 9 introns with a total length of about 60 kb. The transcription start site was determined, and the 5' flanking sequence was analyzed for its potential as a promotor. This region lacks a TATA sequence in the appropriate position relative to the transcription start, is very GC-rich, and contains upstream of the transcription start four Sp1 sites, a feature common to many TATA-less promotors. Based on the homology with DNA binding consensus sequences of transcription factors, we identified in this promotor region several putative DNA binding sites for transcription factors, such as NF-kappa B, Myb, Ets-1, Myc, and ATF. Transfection experiments with a construct containing the PTPA promotor region inserted 5' of a luciferase reporter gene revealed that the 5' flanking sequence of the PTPA gene indeed displayed promotor activity that seems to be cell-line dependent. By fluorescence in situ hybridization and G-banding, the PTPA gene was localized to the 9q34 region. The PTPA gene is positioned centromeric of c-abl in a region embracing several genes implicated in oncogenesis.

Amino Acid Sequence↗

PubMed2000

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

CDC28 Protein Kinase, S cerevisiae↗

PubMed1998

LAR tyrosine phosphatase receptor: a developmental isoform is present in neurites and growth cones and its expression is regional- and cell-specific.

Transgenic mice and Drosophila mutant studies demonstrate that the leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTPase) receptor is required for formation of neural networks. We assessed the hypothesis that alternative splicing of the LAR extracellular region contributes to this function by establishing temporospatial expression patterns of LAR isoforms containing an alternatively spliced extracellular nine amino acid segment (LAR alternatively spliced element-c; LASE-c). LASE-c was present in multiple alternatively spliced and truncated LAR transcripts. In contrast to LAR isoforms without LASE-c, levels of LAR transcripts and protein isoforms containing LASE-c were primarily present during development, suggesting a mechanism for developmental regulation of LAR function. In situ analysis demonstrated increasingly region- and cell-specific expression of LASE-c during maturation. Immunostaining revealed LASE-c-containing LAR protein along neurites and in growth cones. The discovery of highly regulated, temporospatial extracellular domain alternative splicing of LAR-type PTPase receptors points to a novel mechanism by which these receptors might influence network formation.

Animals↗

PubMed1976

Histochemical studies on the distribution of alkaline phosphatase, acid phosphatase, 5-nucleotidase and ATPase in various reproductive tissues of certain digenetic trematodes.

Out of other functions performed by vitellaria in digenetic trematodes, their role in the formation of shell globules and shell membrane of the capsule, as well as in the excretion of iron with the help of vitamin C is very important. The present histochemical work shows the localization of certain enzymes in different parts of the reproductive system of ten species of trematodes viz.: Neopronocephalus triangularis Mehra, 1932; Glossimetra orientalis Mehra, 1937; Orientodiscus lobatus Srivastava, 1938; Eumegacetes artemii Mehra, 1935; Ganeo tigrinus mehra et Negi, 1928; Encyclometra caudata Dollfus, 1928; Thapariella udaipurensis Gupta and Sharma, 1970; Paradistomoides indicum Narain et Das, 1929; Patagifer wesleyi Verma, 1936; Proalarioides tropidonotus Vidyarthi, 1937 and indicates their functional significance. The hydrolytic enzymes (alkaline phosphatase, acid phosphatase, 5-nucleotidase and ATPase) are suggestive of their involvement in the uptake of certain nutrients, glycogen and lipoprotein being very significant among others. The four enzymes could also be detected in testes, ovary, uterus, cirrus sac and egg shell. The possible functional significance of each enzyme has been discussed.

Acid Phosphatase↗

PubMed1978

Alkaline phosphatase and tartrate resistant acid phosphatase activity in cells of prolymphocytic leukemia.

In a typical case of prolymphocytic leukemia, blood smears and lymph node imprints have been investigated cytologically and cytochemically. It could be shown that many leukemic cells in both blood smears and lymph node imprints contained tartrate resistant acid phosphatase activity. Furthermore, the lymph node imprints disclosed many cells with a positive alkaline phosphatase reaction. Such a reaction hitherto has not been described in malignant cells of lymphoproliferative diseases. The cytochemical results underline that prolymphocytic leukemia indeed is a separate entity which can be differentiated from hairy cell leukemia and chronic lymphatic leukemia not only morphologically but also cytochemically. In addition, the case shows that leukemic blood cells are not inevitably identical with those occurring in organ infiltrates.

Acid Phosphatase↗

PubMed2003

Lithium, but not valproate, induces the serine/threonine phosphatase activity of protein phosphatase 2A in the rat brain, without affecting its expression.

Protein phosphatase 2A (PP2A) regulates protein kinase cascades and thus plays an important role in the regulation of cell growth, gene expression and development. We examined the influence of lithium and valproate on the expression of PP2A and its serine/threonine phosphatase activity in the rat frontal cortex and hippocampus. Western blot and immunohistochemical analyses demonstrated that neither lithium nor valproate treatment had an effect on the levels of PP2A immunoreactivity in these brain regions. However, administration of lithium for 1 or 14 days significantly upregulated the activity of PP2A in the frontal cortex. Similarly, lithium administration tended to increase the activity of PP2A in the hippocampus. In contrast, neither a single nor repeated administration of valproate affected the activity of PP2A in these brain regions. These findings indicate that lithium, but not valproate, upregulated the activity of PP2A in the rat brain. It is suggested that the changes in neuronal functions induced by PP2A may be, at least in part, associated with the therapeutic action of lithium.

Animals↗

PubMed2005

An acid phosphatase from Manihot glaziovii as an alternative to alkaline Phosphatase for molecular cloning experiments.

An acid phosphatase, free of deoxyribonuclease activity, was isolated from Manihot glaziovii leaves. It had a Mr of 78 kDa and was optimally active at pH 4.3 and 52 degrees C. It was inactivated at 65 degrees C over 15 min. It had a broad substrate specificity with strongest activity towards p-nitrophenyl phosphate. The enzyme dephosphorylated linearized pUC18 DNA and preventing self-ligation under the same conditions used for calf intestine alkaline phosphatase.

Acid Phosphatase↗

PubMed1995

Participation of a proton-translocating plasma membrane ATPase, acid phosphatase and alkaline phosphatase in ATP degradation by Aspergillus niger extracts.

Extracts of A. niger could catalyze sequential hydrolysis of the three phosphate moieties of the ATP molecule optimally at pH 2 and at pH 8. At pH 2 the hydrolysis was effected by an ATPase followed by acid phosphatase while at pH 8 alkaline phosphatase was the only involved enzyme. Separation of these three phosphate-hydrolyzing enzymes was achieved by Sephadex G-100 column chromatography. The A. niger ATPase seems to have two unique features. First, it was easily solubilized in distilled water and second it had optimum activity at pH 2. The activity of this enzyme was not affected on addition of Na+, K+ or Ca2+ to the assay reaction mixture. It was neither inhibited by sodium azide nor by potassium nitrate but inhibited by orthovanadate, DES, DCCD, Mg2+ and Pi. The substrate concentration-activity relationship was of the hyperbolic type. The enzyme had high specificity for ATP, was inert with ADP and its activity with GTP represented about 6% only of that obtained with equimolar amount of ATP.

Acid Phosphatase↗

PubMed1976

Effects of prednisolone metasulfabenzoate on the induction of DNase II in comparison to alkaline phosphatase and acid phosphatase activities in cultures of HeLa S3 cells.

Cultures of HeLa S3 cells were treated with prednisolone metasulfobenzoate (Na), a derivative of prednisolone which is readily soluble in water. The steroid induced an increase in DNase II, a lysosomal enzyme which was not used previously in enzyme induction by steroids. Alkaline phosphatase, a known inducible enzyme by other steroids and acid phosphatase, a known uninducible enzyme by other steroids, were included for comparative reasons.

Acid Phosphatase↗

PubMed1994

Three genes for protein phosphatase 1 map to different human chromosomes: sequence, expression and gene localisation of protein serine/threonine phosphatase 1 beta (PPP1CB).

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, termed PP1 beta, was isolated from a human teratocarcinoma library. Hybridisation with different cDNA fragments showed that all human tissues examined contained 3.1 kb, 4.0 kb and 5.4 kb PP1 beta mRNAs arising from alternative splicing of the 3' noncoding region. The level of the 5.4 kb mRNA relative to the 3.1 kb mRNA was higher in skeletal muscle than in other tissues and the PP1 beta/PP1 alpha mRNA ratio in rabbit tissues was highest in skeletal muscle. The 3' noncoding region of PP1 beta showed extreme conservation (> or = 90% identity) between man and rodents over 1.7 kb, suggesting that this region is of functional importance. The gene for human PP1 beta (PPP1CB) was localised to chromosome 2 by analysis of somatic cell hybrid DNA and mapped to band q23 by fluorescence in situ hybridization. These data show that the genes for three protein phosphatase catalytic subunits PP1 alpha, PP1 beta, PP1 gamma are all located on different chromosomes.

Amino Acid Sequence↗

PubMed1994

Localization of phosphatase inhibitor-1 mRNA in the developing and adult rat brain in comparison with that of protein phosphatase-1 mRNAs.

The localization and ontogenic changes in the gene expression for phosphatase inhibitor-1 (I-1) were analyzed by in situ hybridization histochemistry, and they were compared with those for three catalytic subunits of protein phosphatase type 1 (PP-1). At the adult stage, intense expression signals for I-1 were detected in the hippocampal formation, piriform cortex, claustrum, dorsal endopiriform cortex, suprachiasmatic nucleus, choroid plexus, arachnoid membrane, and pineal body. Moderate expression signals for I-1 were observed in the olfactory neuronal layers, caudate putamen, layers II-IV, and VI of the neocortex, and cerebellar granule cells, whereas the expression levels were low in the thalamus, cerebellar Purkinje cells, and brain stem nuclei. Although the expression levels for the three PP-1 mRNAs varied notably in various brain regions, a relatively high and parallel expression of I-1 and PP-1 mRNAs was found in most regions of the forebrain. However, the dissociation in the expression levels between I-1 and PP-1 mRNAs was found in several loci: the laminar expression of I-1 mRNA versus the homogeneous expression of PP-1 mRNAs in the cerebral cortex; low levels of expression of I-1 mRNA versus relatively high expression of PP-1 mRNAs in the brain stem nuclei; high expression of I-1 mRNA in the arachnoid membrane versus low expression of PP-1 mRNAs in it. The unparallel expression was also seen in embryonic brain: No significant expression of I-1 mRNA versus substantial expression of PP-1 mRNAs in the ventricular zone and cerebellar external granular layer; transiently high expression of I-1 mRNA in developing thalamus versus constantly moderate to low expression of PP-1 mRNAs there. These findings suggest that I-1 may play some discrete roles independent of the regulation of PP-1 in certain regions and developing stages of the brain.

Animals↗

PubMed1996

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea:Decapoda).

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M(r) 70,000 and M(r) 67,000, as well as less intense bands of proteins with M, 64,000 and M(r) 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M(r) 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl2, CuCl2 and Pb(NH3)3 at millimolar concentrations, but less inhibition was observed with CaCl2.

Alkaline Phosphatase↗

PubMed1986

Evolution of localization of the reactions of adenosine triphosphatase (Mg++-ATP-ase), 5'nucleotidase (5'nt), alkaline phosphatase (AP), and acid phosphatase (AcP) in developing rat testis. I. Physiological conditions.

The experiments were performed upon the rats aged 1, 4, 7, 15, 30, 45, 60, 90 d, and 1,5 a. The behavior of the following reactions was described: for adenosine triphosphatase stimulated by Mg++(Mg++-ATP-ase), for 5'nucleotidase (5'Nt), for alkaline phosphatase (AP), for acid phosphatase (AcP). The first 3 are markers of the transport enzymes in cells, and the 4th is a marker of lytic processes. It was estimated on the basis of the examined reactions that a full metabolic maturity of the gonad was revealed since the 45th d of post-fetal life.

5'-Nucleotidase↗

PubMed1997

Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases.

The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for 18(V/K)bridge, 1.0007 +/- 0.0001 for 15(V/K), and 1.0018 +/- 0.0003 for 18(V/K)nonbridge. The values for 18(V/K)nonbridge and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)bridge of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

Hydrogen-Ion Concentration↗