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PubMed1982

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat.

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.

Alkaline Phosphatase↗
PubMed2001

PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model.

The tumour suppressor gene PTEN/MMAC1/TEP1 encodes a dual-specificity phosphatase that recognizes phosphatidylinositol-3,4,5-triphosphate and protein substrates. We have shown previously that over-expression of PTEN in a tetracycline-controlled inducible system blocks cell cycle progression and induces apoptosis in MCF-7 breast cancer cells. Here, we demonstrate that over-expression of wild-type PTEN leads to the suppression of cell growth through the blockade of cell cycle progression, an increase in the abundance of p27, a decrease in the protein levels of cyclin D1 and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-dead mutant, C124S, promotes cell growth and has the opposite effect on the abundance of p27, cyclin D1 levels and the phosphorylation of Akt. The G129E mutant, which does not have lipid phosphatase activity but retains protein phosphatase activity, behaves like C124S except that the former causes decreases in cyclin D1 levels similar to wild-type PTEN. Therefore, PTEN exerts its growth suppression through lipid phosphatase-dependent and independent activities and most likely, via the coordinate effect of both protein phosphatase and lipid phosphatase activities. Addition of either estrogen or insulin abrogates PTEN-mediated up-regulation of p27 and partially blocks PTEN-mediated growth suppression, whereas the combination of estrogen and insulin eliminates the alterations of p27 and cyclin D1 and completely blocks PTEN-mediated growth suppression. Our findings demonstrate that PTEN blocks cell cycle progression differentially through down-regulating the positive cell cycle regulator, cyclin D1, by its protein phosphatase activity, and up-regulating the negative cell cycle regulator, p27, by its lipid phosphatase activity.

Amino Acid Substitution↗
PubMed1981

Purification and characterization of Mg2+-dependent glycogen synthase phosphatase (phosphoprotein phosphatase IA) from rat liver.

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.

Animals↗
PubMed1981

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities.

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.

Adenosine Triphosphate↗
PubMed1991

Turkey gizzard smooth muscle myosin phosphatase-III is a novel protein phosphatase.

Chromatography of turkey gizzard extract on Sephacryl S-300 has been shown to fractionate the various smooth muscle phosphatases. We have previously reported the purification and characterization of three of these enzymes, termed smooth muscle phosphatase (SMP)-I, -II, and -IV. Recently, we have purified SMP-III to near homogeneity. Although all of the smooth muscle phosphatases dephosphorylate the isolated myosin light chains, only SMP-III and -IV are active toward intact myosin and, therefore, are most likely to play a direct role in the muscle contraction-relaxation process. SMP-III has a higher molecular weight (390,000), as determined by gel filtration, than the other smooth muscle phosphatases and migrates as single band with a molecular weight of 40,000 in a sodium dodecyl sulfate-polyacrylamide gel. SMP-III is immunologically distinct from SMP-I and -II. It dephosphorylates heavy meromyosin and the isolated myosin light chains at a rapid rate but has low activity toward phosphorylase alpha. The activity of SMP-III is not affected by Ca2+ but is activated by Mn2+.Mg2+ stimulates the activity toward heavy meromyosin but inhibits the myosin light chain phosphatase activity. Attempts to classify SMP-III according to the scheme proposed by Ingebritsen and Cohen (Ingebritsen T. S., and Cohen, P. (1983) Science 221, 331-338) revealed that it is resistant to the heat stable inhibitor-2, suggesting that it is a Type 2 protein phosphatase. However, SMP-III is inhibited by concentrations of okadaic acid which are characteristic of Type 1 protein phosphatases and it binds to heparin-Sepharose like other Type 1 phosphatases. But most interestingly, SMP-III does not dephosphorylate the alpha- or beta-subunits of phosphorylase kinase, a property not reported for any Ser/Thr protein phosphatase.

Animals↗
PubMed1993

Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae.

Two protein tyrosine phosphatase genes, PTP1 and PTP2, are known in Saccharomyces cerevisiae. However, the functions of these tyrosine phosphatases are unknown, because mutations in either or both phosphatase genes have no clear phenotypic effects. In this report, we demonstrate that although ptp2 has no obvious phenotype by itself, it has a profound effect on cell growth when combined with mutations in a novel protein phosphatase gene. Using a colony color sectoring assay, we isolated 25 mutants in which the expression of PTP1 or PTP2 is required for growth. Complementation tests of the mutants showed that they have a mutation in one of three genes. Cloning and sequence determination of one of these gene, PTC1, indicated that it encodes a homolog of the mammalian protein serine/threonine phosphatase 2C (PP2C). The amino acid sequence of the PTC1 product is approximately 35% identical to PP2C. Disruption of PTC1 indicated that the PTC1 function is nonessential. In contrast, ptc1 ptp2 double mutants showed a marked growth defect. To examine whether PTC1 encodes an active protein phosphatase, a glutathione S-transferase (GST)-PTC1 fusion gene was constructed and expressed in Escherichia coli. Purified GST-PTC1 fusion protein hydrolyzed a serine phosphorylated substrate in the presence of the divalent cation Mg2+ or Mn2+. GST-PTC1 also had weak (approximately 0.5% of its serine phosphatase activity) protein tyrosine phosphatase activity.

Amino Acid Sequence↗
PubMed1995

Point mutation in the second phosphatase domain of CD45 abrogates tyrosine phosphatase activity.

CD45 is a transmembrane protein tyrosine phosphatase that possesses two phosphatase domains in its cytoplasmic region. Whether both domains function independently as phosphatase enzymes or whether both domains interact to form an active enzyme is unclear. A point mutation of a critical cysteine residue in domain I is known to abolish CD45 activity, implying that the catalytic activity resides in domain I. In this report, mutational analysis of purified, recombinant CD45 cytoplasmic domain protein was performed. It was found that a single amino acid change in domain II (glutamine 1180 to a glycine) resulted in an inactive phosphatase enzyme, whereas two other point mutations in the membrane proximal region of the molecule had no effect on activity. Deletion of the region linking the two phosphatase domains also abolished enzymatic activity. Amino acids crucial for phosphatase activity thus reside in both phosphatase domains of CD45, illustrating that the phosphatase domains of CD45 do not act independently, but are both required for the phosphatase activity of CD45.

Amino Acid Sequence↗
PubMed1982

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase. Use of an exogenous phosphatase to study substrate-mediated regulation of dephosphorylation.

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.

Alkaline Phosphatase↗
PubMed1986

Ontogenic and phylogenic studies of intestinal, hepatic, and placental alkaline phosphatases. Evidence that intestinal alkaline phosphatase is a late evolutionary development.

We have studied the intrauterine development of guinea pig and rat organ phosphatases using biochemical, immunologic, and histologic techniques. In all organs tested the features of the adult phosphatase activity were achieved during the second or third gestational phases. In the rat, the tissue-unspecific phosphatase activity was found by immunoprecipitation with antiserum to rat liver phosphatase in all gestational phases in liver and placenta. The high liver activity in the first phase of gestation corresponded to hematopoietic cells. Hepatocyte phosphatase did not appear until the second gestational phase. The tissue-unspecific phosphatase activity was found in the first and second gestational phase in surface intestinal epithelial cells, even after crypt formation occurred. Once phosphatase appeared in enterocytes on villi, only the intestinal-type enzyme was detected. Alkaline phosphatase was measured in the liver and intestine from animals in various phyla. In fish and reptiles, the intestinal activity had the enzymatic characteristics of the tissue-unspecific enzyme. The appearance of intestinal alkaline phosphatase with unique properties and high specific activity is a characteristic of mammals.

Alkaline Phosphatase↗
PubMed1996

Identity of zinc ion-dependent acid phosphatase from bovine brain and myo-inositol 1-phosphatase.

A 62 kDa Zn(2+)-dependent acid phosphatase has been purified from bovine brain. The protein was carboxymethylated and then cleaved by endoproteinase Glu-C, trypsin and CNBr. Several fragments were subjected to structural analysis either by using mass spectrometry or automated peptide sequencing. The four sequenced peptides were compared with the known protein sequences contained in the EMBL Data Bank. All four peptide sequences were identical to the corresponding amino-acid sequences present in myo-inositol 1-phosphatase from bovine brain. Furthermore we found that the amino-acid composition of Zn(2+)-dependent acid phosphatase purified in our laboratory is very similar to that of myo-inositol 1-phosphatase, and that several peptide fragments have molecular weights (measured by mass spectrometry techniques) identical to those expected for cleavage-fragments originated from the authentic myo-inositol 1-phosphatase. This is one of the key enzymes in the receptor-stimulated inositol phospholipid metabolism and it has been considered as the probable target of Li+ ion during LiCl therapy in manic-depressive patients. The comparison of the Zn(2+)-dependent acid phosphatase and the Mg(2+)-dependent myo-inositol-1-phosphatase activities, measured at different purification steps, shows that the ratio between the two activities was remarkably constant during enzyme purification. We also demonstrated that in the presence of Mg2+ this enzyme efficiently catalyses the hydrolysis of myo-inositol 1-phosphate, and that the Li+ ion inhibits this activity. Furthermore, the thermal treatment of the enzyme causes a time-dependent parallel decrease of both Zn-dependent p-nitrophenyl phosphatase (assayed at pH 5.5) and Mg(2+)-dependent myo-inositol-1-phosphatase (assayed at pH 8.0) activities, suggesting the hypothesis that the same protein possesses both these activities.

Acid Phosphatase↗
PubMed1995

Histone II-A stimulates glucose-6-phosphatase and reveals mannose-6-phosphatase activities without permeabilization of liver microsomes.

The effect of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase activities was investigated in relation to microsomal membrane permeability. It was found that glucose-6-phosphatase activity in histone II-A-pretreated liver microsomes was stimulated to the same extent as in detergent-permeabilized microsomes, and that the substrate specificity of the enzyme for glucose 6-phosphate was lost in histone II-A-pretreated microsomes, as [U-14C]glucose-6-phosphate hydrolysis was inhibited by mannose 6-phosphate and [U-14C]mannose 6-phosphate hydrolysis was increased. The accumulation of [U-14C]glucose from [U-14C]glucose 6-phosphate into untreated microsomes was completely abolished in detergent-treated vesicles, but was increased in histone II-A-treated microsomes, accounting for the increased glucose-6-phosphatase activity, and demonstrating that the microsomal membrane was still intact. The stimulation of glucose-6-phosphatase and mannose-6-phosphatase activities by histone II-A was found to be reversed by EGTA. It is concluded that the effects of histone II-A on glucose-6-phosphatase and mannose-6-phosphatase are not caused by the permeabilization of the microsomal membrane. The measurement of mannose-6-phosphatase latency to evaluate the intactness of the vesicles is therefore inappropriate.

Animals↗
PubMed1976

Insulin stimulation of heart glycogen synthase D phosphatase (protein phosphatase).

Insulin rapidly produced an increase in per cent of total heart glycogen synthase in the I form in fed rats. In fasted rats the response was diminished and delayed. In diabetic animals there was no response over the 15-min time period studied. Since synthase phosphatase activity is necessary for synthase D to I conversion, the phosphatase activity was determined in extracts from these groups of animals. In the fasted and diabetic rats phosphatase activity was less than one-half of that in fed animals. Administration of insulin to fasting animals increased synthase phosphatase activity to a level approaching that of fed animals by 15 min. In diabetic animals insulin also stimulated an increase in synthase phosphatase activity but 30 min were required for full activation. Insulin had no effect in normal fed animals. Insulin activation of synthase phosphatase activity in heart extracts from fasted animals was still present after Sephadex G-25 chromatography and ammonium sulfate precipitation. Thus insulin had induced a stable modification of the phosphatase itself or of its substrate synthase D rendering the latter a more favorable substrate for the reaction. A difference in sensitivity of the reaction to glycogen inhibition was present between fed and fasted animals. Increasing concentrations of glycogen had only a slight inhibitory effect in extracts from fed animals but considerably reduced activity in extracts from fasted animals. Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition. This could explain, at least in part, the increased phosphatase activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.

Animals↗
PubMed1996

Purification and characterization of protein phosphatase 2C in rat parotid acinar cells: two forms of Mg(2+)-activated histone phosphatase and phosphorylation by cAMP-dependent protein kinase.

Two forms of Mg(2+)-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography. Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA. The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak. These two peaks eluted at distinct positions on the gel filtration column. The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS-gels. The first 46-kDa peak immunoreacted with anti-PP2Calpha phosphatase antibody and like PP2Calpha phosphatase could be phosphorylated by cAMP-dependent protein kinase. The second 55-kDa peak showed neither reactivity with anti-PP2Calpha phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+ or Mn2+ dependence for its histone phosphatase activity. Ca2+ showed a strong inhibition on this activity. On the basis of these observations, we have identified the first peak enzyme as PP2Calpha phosphatase and the second peak as a novel PP2C-like phosphatase.

Animals↗
PubMed1987

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A.

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.

Animals↗
PubMed1994

Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay.

Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.

Animals↗
PubMed1993

Phosphatase 2A associated with polyomavirus small-T or middle-T antigen is an okadaic acid-sensitive tyrosyl phosphatase.

Papovavirus tumor antigens have been shown to associate with the cellular phosphoserine/threonine-specific protein phosphatase 2A (PP2A). We were interested in the consequences that T-antigen association might have on PP2A activity and so studies of the phosphatase activity in immunoprecipitates, prepared from polyoma virus-transformed or polyoma virus-infected mouse 3T3 fibroblasts, were performed. The phosphoserine/threonine phosphatase activity, measured with phosphorylase a as the substrate, showed all the characteristics of PP2A. It was stimulated by polycations, inhibited by fluoride or p-nitrophenyl phosphate, sensitive to okadaic acid and microcystin and insensitive to inhibitor-1 and inhibitor-2. Phosphotyrosyl phosphatase (PTPase) activity was associated with the middle-T/small-T-associated complex when reduced, carboxamidomethylated and maleylated lysozyme, phosphorylated exclusively on tyrosyl residues, was used as the substrate. This PTPase activity was as sensitive to okadaic acid as was the phosphorylase phosphatase activity; it could be inhibited by phosphorylase a and did not dephosphorylate poly(Glu80Tyr20). The level of middle-T/small-T-associated PTPase activity relative to the phosphorylase phosphatase activity was tenfold higher than that of the purified dimeric PP2A. A similar activity ratio was observed with the purified phosphatase after stimulation with a cellular protein, designated phosphotyrosyl phosphatase activator. These results suggest that the same enzyme may possess dual specificity. In contrast to the cellular trimeric PP2A, containing the 55-kDa putative regulatory subunit, the middle-T/small-T-associated enzyme had low activity towards a retinoblastoma peptide phosphorylated by p34cdc2. These results indicate how middle-T/small-T might effect the activity of PP2A in polyoma virus-transformed cells.

3T3 Cells↗
PubMed1985

Specificity of protein phosphotyrosine phosphatases. Comparison with mammalian alkaline phosphatase using polypeptide substrates.

The specificity of cytosolic protein phosphotyrosine (PPT) phosphatases was investigated using different peptides and proteins that were phosphorylated on tyrosine residues by the EGF receptor kinase. The acidic phosphoproteins, serum albumin, casein, and myosin light chains, were dephosphorylated by the PPT phosphatases with apparent Km values of 1.2 to 12.5 microM and apparent velocities of 0.2 to 18 mumol/min/mg. In contrast, [Tyr(32P)]histone and the phosphotyrosine peptides [Val5]angiotensin and RR-src, a peptide with sequence Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, were unreactive with the PPT phosphatases. However, each of these unreactive phosphopolypeptides was dephosphorylated under the same conditions by calf-intestine alkaline phosphatase. The data reveal how PPT phosphatase activity has been ascribed to different cellular enzymes. When acidic phosphotyrosine proteins were used as substrates in assays for PPT phosphatase activity the cytosolic enzymes were isolated, whereas when phosphotyrosine histones were used as substrates only the membrane-bound alkaline phosphatase was detected. Apparently the protein tyrosine kinase and the protein tyrosine phosphatases do not have the same specificity, so substrates such as histone, angiotensin, or RR-src are phosphorylated but not hydrolyzed. Therefore, these polypeptides would be ideal for the characterization of protein tyrosine kinases in cellular extracts.

Alkaline Phosphatase↗