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PubMed1988

Component requirements for NADH inhibition and spermine stimulation of pyruvate dehydrogenaseb phosphatase activity.

The subunit and subdomain requirements for NADH inhibition as well as Ca+ and spermine activation of the pyruvate dehydrogenaseb phosphatase were analyzed. The transacetylase-protein X subcomplex (E2-X) was required for all three effects. The oligomeric inner domain of the transacetylase did not support any of these regulatory effects. The presence of at least a portion of the outer (lipoyl-bearing) domains of the transacetylase but not the lipoyl-bearing portion of protein X was essential for expression of these regulatory effects on phosphatase activity. The inner domain of protein X may contribute to some effects. The E2-X subcomplex, alone, had no effect on phosphatase activity in the absence of Ca2+, but the subcomplex did support both NADH inhibition and spermine activation in the absence of Ca2+. Studies with peptide substrates established that spermine is directly bound by a phosphatase subunit. With the resolved pyruvate dehydrogenase component (E1b) used as the substrate, the E2-X subcomplex transformed the effect of spermine from inhibiting to stimulating the rate of dephosphorylation by the phosphatase. The above observations suggest that binding of E1b to the E2-X subcomplex alters its presentation to the phosphatase. We also present several observations that are consistent with NADH inhibition of the phosphatase being mediated through a dihydrolipoyl dehydrogenase-dependent reduction of lipoyl moieties in the E2-X subcomplex. Overall, our data establish that the outer, lipoyl-bearing domains of the oligomeric transacetylase core have an essential role in the function and regulation of the pyruvate dehydrogenase phosphatase.

Alkylation↗
PubMed1985

Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000.

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.

Animals↗
PubMed1980

[Diurnal variation of the elevated acid phosphatase activity in cases of prostate carcinoma (author's transl)].

The diurnal rhythm of total acid phosphatase and prostatic phosphatase activities was investigated in patients with prostate carcinoma. In these patients, the activities of total acid phosphatase, the tartrate-sensitive fraction of acid phosphatase, and lactate dehydrogenase decrease after therapy, whereas the activity of alkaline phosphatase increases. In all patients with prostate carcinoma, the total and tartrate-inhibited acid phosphatase, and the level of cortisol show a diurnal rhythm before therapy, with a minimum at night. In one patient, after orchiectomy, the cortisol rhythm remained unchanged, but the daily phosphatase variation was absent. Diurnal variations of lactate dehydrogenase and alkaline phosphatase were also observed in 2 patients without prostate carcinoma, but with elevated levels of these enzymes.

Acid Phosphatase↗
PubMed1995

Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.

Alkaline Phosphatase↗
PubMedDate not supplied

The mechanism of elevated alkaline phosphatase activity after bile duct ligation in the rat.

Alkaline phosphatase activity in the liver and intestine increases after bile duct ligation, reportedly by increased enzyme synthesis. To ascertain the mechanism of this increased synthesis in the absence of a cDNA clone encoding the enzyme, we have estimated the concentration of liver and intestinal alkaline phosphatase mRNA by translational analysis. Monospecific antiserum to rat placental alkaline phosphatase was raised. The resulting antiserum precipitated two peptides of 53 and 56 kd after translation of liver poly(A) + RNA. The precipitation of both peptides was blocked by the single 64 kd placental alkaline phosphatase. Processing of the cell-free products by microsomal membranes produced peptides of 62 and 64 kd. Antiserum to rat intestinal alkaline phosphatase also identified two peptides as products of intestinal RNA translation. After bile duct ligation, we confirmed a transient 2-fold increase in alkaline phosphatase activity in the intestine and a more constant 7-fold increase in the liver. However, the alkaline phosphatase mRNA concentration remained unchanged in both organs. We conclude that increased alkaline phosphatase synthesis after bile duct ligation results from an enhanced rate of translation of mRNA.

Alkaline Phosphatase↗
PubMed1993

Differential expression of alkaline phosphatase in clones of human osteoblast-like cells.

We established cultures of cells growing out from adult bone chips and maintained them through 12 passages in culture. The cultures showed osteoblastic phenotype accompanied by synthesis of collagen type I, osteonectin, alkaline phosphatase, and osteocalcin. We report the characterization of 21 clones obtained from three different individual primary cultures. We studied the expression of osteonectin, alkaline phosphatase, collagen, and osteocalcin in the clones. Metabolic labeling showed production of type I collagen and of osteonectin in all clones studied. In two-thirds of the clones and in mass cultures alkaline phosphatase was not detected at passage 2, but it was detected in increasing amounts at later passages in culture. The clones attained different but detectable levels of expression of this marker by passage 8. The different levels in the expression of alkaline phosphatase in positive clones may be because they were derived from cells at different stages of osteoblastic maturation or due to small changes in microenvironment. The alkaline phosphatase-positive clones were tested for osteocalcin, and they showed measurable expression only at passage 10. A third of the clones obtained were negative for alkaline phosphatase during 12 passages in culture. The obtainment of clones unable to produce alkaline phosphatase may be due to loss of differentiating potential under the in vitro culture conditions. The growth rate and potential of all clones studied were similar through 12 passages in culture, regardless of their potential for expression of alkaline phosphatase.

Adult↗
PubMed2001

Suppressive role of endogenous regucalcin in the regulation of protein phosphatase activity in rat renal cortex cytosol.

The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.

Animals↗
PubMedDate not supplied

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex.

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.

Animals↗
PubMed1995

High-level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae.

The secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid-encoded acid phosphatase, a secreted endogenous glycoprotein. A 2-microns-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest. This is not per se due to leucine starvation, but due to intracellular accumulation of highly glycosylated enzymatically active acid phosphatase. Immunofluorescence and cytological analysis indicate that intracellular accumulation of acid phosphatase occurs in a subpopulation of cells. By Ludox-AM density centrifugation, these cells can be enriched on the basis of their higher density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non-accumulating cells of low density. These cells are defective in directed secretion and bud formation, therefore can no longer grow and show dramatic changes in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in growth and to the intracellular accumulation of further expressed acid phosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secretion at this stage.

Acid Phosphatase↗
PubMed1998

Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.

Acid Phosphatase↗
PubMed1995

Trypanosoma brucei and Trypanosoma cruzi: life cycle-regulated protein tyrosine phosphatase activity.

Recent evidence that Trypanosoma brucei synthesizes stage-regulated phosphotyrosine containing proteins and protein kinases stimulated us to assay bloodstream and insect stages of Trypanosoma cruzi and both pleomorphic and monomorphic clones of T. brucei for tyrosine phosphatase activity. Bloodstream and procyclic insect stages of T. brucei contained a 55-kDa protein that cross-reacted with monoclonal antibodies directed against the human placental tyrosine phosphatase PTP1B. Protein lysates of all life cycle stages of both trypanosomes dephosphorylated a nonspecific substrate, pNPP, and the specific substrate Tyr(P)Raytide. Dephosphorylation of Tyr(P)Raytide was effectively inhibited only by sodium vanadate, a specific phosphotyrosine phosphatase inhibitor, but pNPP activity was also inhibited by sodium fluoride (NaF) in lysates of T. brucei and by NaF and sodium tartrate in lysates of T. cruzi, suggesting that their respective lysates also contained serine/threonine and acid phosphatase activities. Fractionation studies revealed that most of this activity was in the cytosol. Stage regulation of tyrosine phosphatase activity in T. cruzi was strongly suggested by differences in the optimal pH for tyrosine phosphatase activity (7.0 for amastigotes and epimastigotes; 5.0 for trypomastigotes). We conclude that both species of trypanosomes synthesize tyrosine phosphatases and propose that identification and characterization of the enzymes responsible for this phosphatase activity could provide information about trypanosomal virulence or the regulation of trypanosomal growth and differentiation.

Animals↗
PubMed1996

Localisation of acid phosphatase activity on the surface of bloodstream forms of Trypanosoma congolense.

In vitro, living bloodstream forms of Trypanosoma congolense were shown to hydrolyse p-nitrophenyl phosphate, a substrate for phosphatases. This activity appears to be from an acid phosphatase because it was enhanced at low pH values, was inhibited by the acid phosphatase inhibitor sodium fluoride, and was not inhibited by the alkaline phosphatase inhibitor tetramisole. The activity did not appear to be secreted into the surrounding medium by the living parasites although phosphatase activity could be detected in the surrounding medium when dead or dying parasites were present. Studies at various temperatures indicated that at least some of this acid phosphatase activity may be associated with the surface of the parasites, rather than with endocytic or intracellular systems. This was supported by subcellular fractionation of radiolabelled parasites which showed some cosedimentation of acid phosphatase activity with radiolabelled iodine. Histochemical studies of the parasites also supported this conclusion. Electron microscopical examination of trypanosomes incubated with lead nitrate and p-nitrophenyl phosphate showed lead phosphate deposits on the surface of the parasites in addition to the expected localisation in the flagellar pocket. We conclude that Trypanosoma congolense possesses a surface-bound acid phosphatase.

Acid Phosphatase↗
PubMed1994

Phosphatase-negative mutants of Legionella pneumophila and their behavior in mammalian cell infection.

Microbial phosphatases are known or suspected to play a role in the pathogenesis of several intracellular pathogens, including Legionella micdadei. Legionella pneumophila also possess phosphatase activities, but their possible roles in cellular infection are unknown. We generated mutants of a serogroup 1 isolate of L. pneumophila that lack the major phosphatase. Isolation of a Pho- mutant after random mutagenesis with transposon MudII4041 allowed us to dissociate the major alkaline phosphatase (pH optimum approximately 8) from a minor acid phosphatase activity. Both activities were concentrated in the bacterial periplasm. The gene encoding the major alkaline phosphatase (pho) was cloned by expression in E. coli and used to generate a site directed mutation in two L. pneumophila strains. Each parent-mutant pair was compared in a U937 cell tissue culture assay for capacity to infect, lyse, and grow within mammalian cells. Although the parental stains differed in their U937 cell cytopathicity, neither was significantly more infective than its Pho- derivative, suggesting that the alkaline phosphatase activity is not essential for cellular infection. Because they are not attenuated, Pho- mutants can be used to generate gene fusions with E. coli alkaline phosphatase to study and secretion and cellular infectivity in L. pneumophila.

Alkaline Phosphatase↗
PubMed1993

Bacterial and viral protein tyrosine phosphatases.

Unrestricted protein tyrosine phosphatase (PTPase) activity may play a role in pathogenesis. For instance, the virulence determinant gene, yopH, of Yersinia pseudotuberculosis encodes a PTPase. The phosphatase activity of the YopH protein is essential for the pathogenesis of Y. pseudotuberculosis. Yersinia pestis, the bacterium which causes the bubonic plague, also contains a gene closely related to yopH. The action of YopH on host proteins appears to break down signal transduction mechanisms in many cell types including those of the immune system. This may contribute to the ability of the bacterium to escape effective surveillance by the immune system. The vaccinia virus VH1 gene, like yopH in the Yersinia bacteria, encodes a protein phosphatase. The VH1 PTPase defines a new class of phosphatases capable of dephosphorylating both phosphoserine/threonine and tyrosine containing substrates. Proteins sharing sequence identity to this dual-specificity phosphatase have been identified from other viruses, yeast and man. Although a complete understanding of the function of these dual-specificity phosphatases is not presently available, they clearly play important roles in cell cycle regulation, growth control and mitogenic signaling mechanisms. The unique catalytic properties of the dual specificity phosphatases suggest that these catalysts constitute a distinct subfamily of phosphatases.

Amino Acid Sequence↗
PubMed1982

Inactivation and reactivation of phosphoprotein phosphatase.

The catalytic subunit of phosphoprotein phosphatase (Mr = 35,000) is inactivated by phosphate compounds such as trimetaphosphate, PPi, and ATP. The inactivation of phosphoprotein phosphatase by these phosphate compounds is time- and concentration-dependent, is not reversed by dilution or gel filtration and is protected by Pi. A dissociation constant for the enzyme-trimetaphosphate complex and a rate constant for the reaction were calculated to be 4.6 x 10(-4) M and 0.29 min-1, respectively. The inactivation of phosphatase by PPi and ATP shows more complex kinetics than that by trimetaphosphate. The addition of EDTA to PPi and ATP exhibits more potent inactivation, even though EDTA alone does not inactivate phosphatase. This phosphoprotein phosphatase is not labeled by [gamma-32P]ATP. The inactivation of phosphatase by PPi or ATP can only be reversed by Mn2+ or Co2+, among all other metals or cationic compounds tried. The reactivation also requires sulfhydryl compounds. The effectiveness of sulfhydryl compounds follows the order: dithioerythritol greater than mercaptoethanol greater than cysteine. Glutathione was without effect. Metal analysis of the catalytic subunit did not reveal any significant amounts of Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Sn, or Zn. Phosphoprotein phosphatase activity from zinc-deficient rat livers also eliminated the possibility of this phosphatase being a zinc metalloenzyme. Inactivation does not seem to be due to a loss of a critical metal ion. Other mechanisms for inactivation are presented.

Animals↗
PubMed1989

Autonomic control of acid phosphatase exocrine secretion by the rat prostate.

In vivo prostatic secretion was collected from retired breeder Sprague Dawley rats using a method for isolated perfusion of the rat prostatic urethra. Enzymatic acid phosphatase determination was performed on the collected effluent. Control acid phosphatase secretion was 24.2 +/- 2.7 nm over 30 minutes. Intravenous phenylephrine 5 mg/kg stimulated a 10 fold increase in acid phosphatase secretion. The secretion seen with phenylephrine was dose dependent and could be blocked with prazosin, but not yohimbine, atropine, or propranolol. Intravenous beta-adrenergic agonist isoproterenol caused no increase in the secretion of rat prostatic acid phosphatase. Intravenous administration of the cholinergic agonist pilocarpine also resulted in a dose dependent rise in acid phosphatase secretion. The stimulation seen could be blocked by atropine but not phentolamine or propranolol. The stimulation of acid phosphatase secretion seen with alpha 1 adrenergic or cholinergic agonists was not additive. Intravenous vasoactive intestinal peptide did not stimulate acid phosphatase secretion nor did it augment the secretion induced by alpha 1 adrenergic or cholinergic agonists. Release of acid phosphatase into rat prostatic exocrine secretion is under both alpha 1 adrenergic and cholinergic control.

Acid Phosphatase↗
PubMed1985

Rat hepatic microsomal glucose-6-phosphatase protein levels are increased in streptozotocin-induced diabetes.

Hepatic microsomal glucose-6-phosphatase activity levels and the hepatic output of glucose are increased in diabetes. We have used protein chemistry and immunological techniques to determine the mechanism by which the activity levels of the glucose-6-phosphatase system are increased in streptozotocin-induced diabetic rats. In the streptozotocin-induced diabetic rats, the activity of the glucose-6-phosphatase enzyme increased four-fold without appreciably altering the transport capacity of the glucose-6-phosphatase system. The solubilized diabetic rat liver glucose-6-phosphatase enzyme appeared to be very similar to the solubilized enzyme from control rat liver microsomes. They exhibit the same Km, are labile at 30 degrees C, are stabilized by sodium fluoride and they migrate to the same position during density gradient centrifugation. Immunological studies demonstrated that a greater amount of hepatic microsomal glucose-6-phosphatase enzyme protein is present in diabetic rats than in control rats. Thus, we have determined for the first time that increased levels of the glucose-6-phosphatase protein are present in streptozotocin-induced diabetes. The significance of this finding in relation to the regulation of the hepatic microsomal glucose-6-phosphatase system is discussed.

Animals↗
PubMed2004

Role of zinc in regulation of protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells: zinc modulation of insulin-like growth factor-I's effect.

Zinc, an essential trace element, has been demonstrated to stimulate bone growth in animal and human. The cellular mechanism by which zinc stimulates bone growth has not been fully clarified. The effect of hormone and zinc on protein tyrosine phosphatase activity in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers, and then exchanged to culture medium containing either vehicle, zinc sulfate or various hormones in the absence of 10% FBS. After medium change, cells were cultured for 48 h. Protein tyrosine phosphatase activity in the lysate of cells was significantly increased by culture with zinc (10(-6) - 10(-4) M). The effect of zinc in increasing the enzyme activity was completely blocked by culture with cycloheximide (10(-7 )M), an inhibitor of protein synthesis, or 5, 6-dichloro-l-beta-D- riboifuranosylbenzimidarzole (DRB) (10(-6) M), an inhibitor of translational activity. Addition of calcium chloride (10 microM) into the reaction mixture caused a significant increase in protein tyrosine phosphatase activity; this increase was completely blocked in the presence of trifluoperazine (50 microM), an antagonist of calmodulin. Culture with zinc caused a significant increase in Ca2+/calmodulin-dependent protein tyrosine phosphatase activity in osteoblastic cells. Protein tyrosine phosphatase activity was significantly raised by culture with parathyroid hormone (human PTH [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]; 10(-7) M), 17beta-estradiol (10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M) or insulin (10(-8) M). The enzyme activity was not significantly enhanced by the addition of calcium (10 microM) into the reaction mixture. The effect of PTH or IGF-I in increasing protein tyrosine phosphatase activity was completely blocked by culture with DRB. The IGF-I-induced increase in enzyme activity was significantly enhanced by culture with zinc. Such an effect was not seen in the case of PTH. Moreover, the effect of IGF-I in increasing proliferation of osteoblastic cells was significantly enhanced by culture with zinc. The effect of PTH was not enhanced by zinc. This study demonstrates that protein tyrosine phosphatase activity in osteoblastic cells is enhanced by various bone anabolic factors, and that zinc modulates the effect of IGF-I on protein tyrosine phosphatase activity and cell proliferation.

3T3 Cells↗