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PubMed1984

Alkaline phosphatase activity in non-Hodgkin's lymphomas and pseudolymphomas of the skin.

Alkaline phosphatase enzyme activity was studied histochemically in 60 non-Hodgkin lymphomas and 10 pseudolymphomas of the skin. Among the 37 B-cell lymphomas, membrane-bound alkaline phosphatase activity was demonstrated in 8 cases. In none of the 23 cutaneous T-cell lymphomas studied could membrane-bound alkaline phosphatase be detected. Among the pseudolymphomas, 2 cases revealed alkaline phosphatase activity. It was not possible to draw any particular clinically significant conclusions from the membrane-bound alkaline phosphatase reactions. Looking for the microenvironmental conditions of lymphoproliferative processes in the skin, alkaline phosphatase-positive capillaries were seen predominantly in the T-cell lymphomas. The stromal reaction showing a proliferation of alkaline phosphatase-positive fibroblasts was more pronounced in cutaneous B-cell lymphomas. In conclusion, membrane-bound alkaline phosphatase in lymphoproliferative processes in the skin, as in the lymph node, characterize a distinct group of B lymphocytes related to follicle center cells. The clinical relevance of this finding remains to be determined.

Alkaline Phosphatase↗
PubMed1998

Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation.

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 mumol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.

Alkaline Phosphatase↗
PubMed1968

Production of phosphatase by Aspergillus awamori var. kawachii in a low phosphate medium.

The effect of phosphate on the production of phosphatases by Aspergillus awamori var. kawachii was studied. In a high phosphate medium, little phosphatase was produced, and the phosphatase activity was predominately for beta-glycerophosphate. In a low phosphate medium, the production of phosphatase was increased and activity for glucose-6-phosphate predominated. Medium containing 1 mg of phosphorus per 100 ml was optimal, and the amount of phosphatase produced in this medium was about 200 times that produced in a high phosphate medium. By means of column chromatography on diethylaminoethyl cellulose, the phosphatase produced in the high phosphate medium was found to be eluted mainly at fraction e; the phosphatase of the low phosphate medium was separated into fractions a, b, c, and d. Thus, the phosphatase fractions produced in the low phosphate medium were different from those of the high phosphate medium. Since no specific effect on the production of esterases was observed when various phosphate esters were used as substrates, the enzymes of phosphate metabolism appear to be activated by nonspecific phosphate sources.

6-Phytase↗
PubMed1971

Interactions of alkaline phosphatase and the cell wall of Pseudomonas aeruginosa.

Spheroplasts prepared by lysozyme treatment of cells of Pseudomonas aeruginosa, suspended in 20% sucrose or 0.2 m MgCl(2), were examined in detail. Preparation of spheroplasts in the presence of 0.2 m Mg(2+) released periplasmic alkaline phosphatase, whereas preparation in the presence of 20% sucrose did not, even though untreated cells released phosphatase when suspended in sucrose in the absence of lysozyme. Biochemical characterizations of the sucrose-lysozyme preparations indicated that lysozyme mediated a reassociation of the released phosphatase with the spheroplasts. In addition, the enzyme released from whole cells suspended in 20% sucrose (which represents 20 to 40% of the cell-bound phosphatase) reassociates with the cells in the presence of lysozyme. Electron microscopic examinations of various preparations revealed that phosphatase released in sucrose reassociated with the external cell wall layers in the presence of lysozyme, that sucrose-lysozyme prepared spheroplasts did not dissociate phosphatase which remained in the periplasm of sucrose-washed cells, and that phosphatase was never observed to be associated with the cytoplasmic membrane. A model to account for the binding of P. aeruginosa alkaline phosphatase to the internal portion of the tripartite layer of the cell wall rather than to the cytoplasmic membrane or peptidoglycan layer is presented.

Alkaline Phosphatase↗
PubMed1974

Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase.

Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [(35)S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.

Alkaline Phosphatase↗
PubMed1997

Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.

EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine. In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine. These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation. Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.

Bacterial Outer Membrane Proteins↗
PubMed1995

A variant alkaline phosphatase found in a case of gastric carcinoma with super bone scan.

A rare case of gastric carcinoma associated with increased serum variant alkaline phosphatase activities is presented. A 54 year old man had extremely high serum alkaline phosphatase activity (18,607 U/l) with normal calcium and phosphate concentrations. His bone scintigram showed abnormal findings, 'super bone scan'. He was diagnosed as having Borrmann type 4 gastric carcinoma with diffuse bone metastases by examinations of the upper gastrointestinal tract and iliac bone biopsy. The alkaline phosphatase isozyme of this patient was of the bone type as measured by cellulose acetate membrane electrophoresis and the placenta/bone type by agarose gel electrophoresis, respectively. Immunoelectrophoresis and the immunoprecipitation method using monoclonal antibodies against various alkaline phosphatase isozymes, however, showed that his serum alkaline phosphatase had the liver type antigenicity. Furthermore, it had a larger molecular size and different sugar chains compared with the common liver type alkaline phosphatase. These findings suggest that a unique variant alkaline phosphatase was produced by gastric cancer cells, which is possibly an explanation for the high serum alkaline phosphatase activities in this patient.

Alkaline Phosphatase↗
PubMed1987

Phosphate transport and alkaline phosphatase in confluent MDCK cell monolayers.

Several studies have suggested that the brush border membrane alkaline phosphatase is involved in the renal reabsorption of inorganic phosphate along the proximal convoluted tubule. However, other studies on the influence of l(-)bromotetramisole, an inhibitor of alkaline phosphatase, upon phosphate transport into brush border membrane vesicles have failed to show an involvement of alkaline phosphatase. The present experiments were designed to demonstrate that the MDCK (Madin, Darby, canine, kidney) cell line can be used as an alternative model to study phosphate transport, to examine the effect of l(-)bromotetramisole and the role of alkaline phosphatase. MDCK cell monolayers were concomitantly used for alkaline phosphatase activity measurement and phosphate transport analysis. While alkaline phosphatase activity increases by 37-fold from day 2 to day 8 of culture, reaching a plateau at day 10, the sodium-dependent phosphate transport into the cell monolayers decreases by 2-fold during that same period. The phosphate transport was also studied in the presence of l(-)bromotetramisole at pH 8.5. The sodium-dependent phosphate uptake of inorganic phosphate is reduced by 43% in the presence of l(-)bromotetramisole at day 3 of culture but is not reduced in the 7-day-old culture. Our results suggest a participation of alkaline phosphatase upon phosphate transport in MDCK cell monolayers and indicate that this cell line constitutes a good model to study the relationship between alkaline phosphatase and phosphate transport.

Alkaline Phosphatase↗
PubMed1979

Prognostic value of bone marrow acid phosphatase in prostatic carcinoma.

The total and l-tartrate labile acid phosphatase were studied in 25 patients with carcinoma of prostate. The results were compared with the results from a control group. Serum acid phosphatase levels both in the control groups and in patients with prostatic cancer were lower than bone marrow acid phosphatase levels. This may be due to acid phosphatase released from blood cells during hemolysis. A positive correlation between serum and bone marrow acid phosphatase levels in patients with prostatic carcinoma was found. There was a significant rise in bone marrow acid phosphatase levels (above the normal range from the control group) in patients with advanced stage III and stage IV prostatic carcinoma with significantly increased serum levels. The levels of bone marrow acid phosphatase gave a supplementary diagnostic method in the diagnosis of prostatic carcinoma. A hypothesis that raised levels of bone marrow acid phosphatase are diagnostic of early metastasis from prostatic carcinoma is given.

Acid Phosphatase↗
PubMed2004

Post-translational generation of constitutively active cores from larger phosphatases in the malaria parasite, Plasmodium falciparum: implications for proteomics.

BACKGROUND: Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue in selected protein phosphatases of the protease-rich malaria parasite, Plasmodium falciparum. RESULTS: P. falciparum encodes a number of Ser/Thr protein phosphatases (PP) whose catalytic subunits are composed of a catalytic core and accessory domains essential for regulation of the catalytic activity. Two examples of such regulatory domains are found in the Ca+2-regulated phosphatases, PP7 and PP2B (calcineurin). The EF-hand domains of PP7 and the calmodulin-binding domain of PP2B are essential for stimulation of the phosphatase activity by Ca+2. We present biochemical evidence that P. falciparum generates these full-length phosphatases as well as their catalytic cores, most likely as intermediates of a proteolytic degradation pathway. While the full-length phosphatases are activated by Ca+2, the processed cores are constitutively active and either less responsive or unresponsive to Ca+2. The processing is extremely rapid, specific, and occurs in vivo. CONCLUSIONS: Post-translational cleavage efficiently degrades complex full-length phosphatases in P. falciparum. In the course of such degradation, enzymatically active catalytic cores are produced as relatively stable intermediates. The universality of such proteolysis in other phosphatases or other multi-domain proteins and its potential impact on the overall proteome of a cell merits further investigation.

Animals↗
PubMed1999

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with spontaneous calcium oscillations and enhanced calcium responses following endothelin-1 stimulation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the signalling molecules inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P4) and thereby regulates cellular transformation. To investigate the role Ins(1,4,5)P3-mediated Ca2+ oscillations play in cellular transformation, we studied Ins(1,4,5)P3-mediated Ca2+ responses in cells underexpressing the 43 kDa 5-phosphatase. Chronic reduction in 43 kDa 5-phosphatase enzyme activity resulted in a 2.6-fold increase in the resting Ins(1,4,5)P3 concentration and a 4.1-fold increase in basal intracellular Ca2+. The increased Ins(1,4,5)P3 levels resulted in partial emptying (40%) of the Ins(1,4,5)P3-sensitive Ca2+ store, however, store-operated Ca2+ influx remained unchanged. In addition, Ins(1,4,5)P3 receptors were chronically down-regulated in unstimulated cells, as shown by a 53% reduction in [3H]Ins(1,4,5)P3 binding to microsomal receptor sites. Agonist stimulation with endothelin-1 resulted in the rapid rise and fall of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels, with no significant differences in the rates of hydrolysis of these second messengers in antisense- or vector-transfected cells. These studies indicate, in contrast to its predicted action, the 43 kDa 5-phosphatase does not metabolise Ins(1, 4,5)P3 and Ins(1,3,4,5)P4 post agonist stimulation. Cells with decreased 43 kDa 5-phosphatase activity exhibited spontaneous Ca2+ oscillations in the absence of any agonist stimulation, and increased sensitivity and amplitude of intracellular Ca2+ responses to both high and low dose endothelin-1 stimulation. We conclude the 43 kDa 5-phosphatase exerts a profound influence on Ins(1,4, 5)P3-induced Ca2+ spiking, both in the unstimulated cell and following agonist stimulation. We propose the enhanced Ca2+ oscillations may mediate cellular transformation in cells underexpressing the 43 kDa 5-phosphatase.

Animals↗
PubMed1976

Alkaline phosphatase of human and calf small intestine. Purification and immunochemical characterization.

Alkaline phosphatase from human and calf small intestines has been prepared and purified until homogeneous, as judged by polyacrylamide gel electrophoresis, by means of the following techniques: n-butanol extraction, ammonium sulfate precipitation, acetone fractionation, ion exchange chromatography and isoelectric focusing in a sucrose density gradient. Three and two alkaline phosphatase froms from human and calf small intestines, respectively could be isolated by preparative isolectric focusing. The relative amounts of these components are not constant, but they have the same catalytic properties, suggesting that they may embody a common protein core with an identical active centre(s). Precipitating antisera for alkaline phosphatase from human and calf intestine have been prepard in rabbits by intramuscular, dermal, subcutaneous and intravenous administration of the pure major component of each enzyme species. Both antisera precipitate completely their homologous as well as their heterologous antigens (intestinal enzyme) and showed partial identity with placental alkaline phosphatase. There was no reaction with alkaline phosphatase from bone, liver heart, spleen, lung, stomach, pancreas, brain, bile-gall bladder and erythrocytes. Alkaline phosphatase preparation from human kidney contains a minor component of the intestinal type, beside many multiple forms which, on treatment with neuramindase, became identical in their electrophoretic and biochemical properties. At least eight multiple forms of the placental enzyme could be shown by isoelectric focusing in polyacrylamide gel. On treatment with neuraminidase these forms became less charged and only four forms remained. All forms were immunologically identical using either anti placental-enzyme or anti intestinal-AP serum. Monospecific antisera against human or calf intestinal alkaline phosphatase were obtained by absorption with purified placental enzyme. This monospecific anti intestinal-AP serum could be used for an immunological quantitative determination of the intestinal isoenzyme in sera or other liquids in the presence of other alkaline phosphatase isoenzymes.

Alkaline Phosphatase↗
PubMed1989

Targeting of lysosomal acid phosphatase with altered carbohydrate.

Human lysosomal acid phosphatase is transported as a transmembrane protein to lysosomes, where it is converted into a soluble protein by a limited proteolysis (Waheed et al., 1988, EMBO J. 7, 2351-2358). Transport of human lysosomal acid phosphatase in heterologous BHK-21 cells was examined under conditions that impair mannose-6-phosphate receptor-dependent transport, N-glycosylation or processing of N-linked oligosaccharides. Targeting of lysosomal acid phosphatase to lysosomes was neither affected by antibodies blocking the mannose-6-phosphate/IGF II receptor, nor by NH4Cl, which inhibited the mannose-6-phosphate receptor-dependent targeting of soluble lysosomal enzymes. 1-Deoxynojirimycin, 1-deoxymannojirimycin and swainsonine inhibited processing of N-linked oligosaccharides in lysosomal acid phosphatase without significantly affecting its transport. Tunicamycin inhibited N-glycosylation of lysosomal acid phosphatase. The non-glycosylated lysosomal acid phosphatase polypeptides accumulated within light membranes and were not transported to dense lysosomes. These results indicate that transport of lysosomal acid phosphatase is independent of mannose-6-phosphate receptors, does not involve an acid pH-dependent step and does not require processing of N-linked oligosaccharides. N-glycosylation appears to be necessary to achieve a transport competent form of lysosomal acid phosphatase.

1-Deoxynojirimycin↗
PubMed2001

Phosphatase activities of rat intestinal enterocytes and their relation to diverse luminal pH, with special references to the possible localization of phytase along the brush border membrane.

Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fluctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chelation although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalanine, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions--which were dependent on Mg++ and stimulated by Ca++ and resistant to L(+)phenylalanine--to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH. These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg++/ Ca++-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.

6-Phytase↗
PubMed1999

Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei.

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl(2) (10(-5) and 10(-4) M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 microM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10(-6)-10(-4) M), an inhibitor of Ca(2+)/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca(2+)/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 microM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei.

Animals↗
PubMedDate not supplied

[Localization of alkaline phosphatase in cells of Bacillus intermedius].

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.

Alkaline Phosphatase↗
PubMed1992

Phosphatase inhibitors modulate the growth-regulatory effects of human tumor necrosis factor on tumor and normal cells.

Tumor necrosis factor alpha (TNF-alpha) has been shown to inhibit the growth of tumor cells and stimulate the growth of certain normal cells in vitro. The mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we investigated the effects of phosphatase inhibitors on the cell growth-inhibitory effects of TNF on L-929, a highly sensitive murine connective tissue tumor cell line, and on the growth-stimulatory effects of TNF on normal human fibroblasts. The antiproliferative effects of TNF on L-929 cells were inhibited by orthovanadate, an inhibitor of phosphotyrosine phosphatases, in a dose-dependent manner. Okadaic acid, which is a specific inhibitor of phosphoserine- and phosphothreonine-specific phosphatases, also blocked the growth-inhibitory effects of TNF, suggesting that TNF may function through the activation of certain phosphatases. These inhibitors had no effect on TNF receptors. Addition of phosphatase inhibitor, even 12 h after the treatment of cells with TNF, was sufficient to block the antiproliferative effects of the cytokine, suggesting that the inhibitor is acting at a late event in the pathway of action of TNF. Cells were protected by orthovanadate from the cytotoxic effects of TNF even in the presence of actinomycin D or cycloheximide, thus indicating the lack of a requirement for de novo protein synthesis. Orthovanadate altered the cell morphology from flat spindle shapes to rounded ones. Besides anticellular effects, a phosphatase inhibitor also suppressed the proliferative effects of TNF on human fibroblasts. These results thus suggest that phosphatases may be needed for both proliferative and antiproliferative effects of this cytokine. This is the first report to suggest that phosphatases play a role in the growth-regulatory action of TNF.

Animals↗
PubMed2004

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats.

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.

Animals↗