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PubMed1990

Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate inhibit inositol-1,3,4,5-tetrakisphosphate 3-phosphatase in rat parotid glands.

In assays containing a physiological concentration of inositol 1,3,4,5-tetrakisphosphate (1 microM), this isomer was attacked by both 3- and 5-phosphatases present in rat parotid homogenates and 100,000 X g supernatant and particulate fractions. As the concentration of cytosolic protein in the assay was decreased, the specific activity of the soluble 3-phosphatase increased significantly. In contrast, the specific activity of particulate 3-phosphatase was independent of protein concentration. At the lowest protein concentrations tested, the sum of soluble and particulate 3-phosphatase specific activities was 2.5-fold greater than that of the parent homogenate. These observations indicate that parotid cytosol contains a hitherto undescribed endogenous mechanism for inhibiting 3-phosphatase. The effects upon 3- and 5-phosphatase of a number of inositol polyphosphates were studied. Both activities were inhibited by inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate (IC50 approximately 50 microM). Inositol 3,4,5,6-tetrakisphosphate was a more potent inhibitor of 3-phosphatase (IC50 about 10 microM) and did not affect 5-phosphatase. Inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate were very potent inhibitors of 3-phosphatase (IC50 values of 1 and 0.5 microM, respectively); these polyphosphates did not affect 5-phosphatase activity at concentrations of up to 10 microM. Inositol 1,3,4,5,6-pentakisphosphate was a competitive inhibitor of the 3-phosphatase, whereas inositol hexakisphosphate was a mixed inhibitor. These data lead to the proposal that the inositol 1,3,4,5-tetrakisphosphate 3-phosphatase is unlikely to be an important enzyme activity in vivo.

Animals↗
PubMed1990

Molecular cloning of complementary DNAs encoding alkaline phosphatase in human colon cancer cells.

We have isolated a set of complementary DNA (cDNA) clones that together encode the alkaline phosphatase of human colon cancer LS174T cells. These clones include two cDNAs isolated from a conventionally prepared oligodeoxythymidylate-primed lambda ZAP cDNA library and three cDNA clones prepared by using the polymerase chain reaction. The deduced amino acid sequence of the alkaline phosphatase primary transcript contains 532 amino acids. This enzyme is similar to, but not identical with, placental alkaline phosphatase (PLAP); it exhibits 12-19 amino acid substitutions when compared to the various alleles of PLAP. Also, it is similar to PLAP in that it is apparently attached to the cell membrane by a phosphatidylinositol-containing anchor as judged by the ability of phosphatidylinositol-specific phospholipase C to release it from membranes. It is different from PLAP however, in terms of its signal sequence which only contains 19 amino acids as compared to 22 for PLAP. Moreover, the 3'-untranslated region of the LS174T cell alkaline phosphatase message diverges considerably from the PLAP message. The LS174T cell alkaline phosphatase cDNAs are actually much more similar to the "germ cell" alkaline phosphatase gene than they are to PLAP. Only 7 amino acid substitutions exist between the LS174T cell enzyme and the alkaline phosphatase encoded by the germ cell alkaline phosphatase genomic DNA clone isolated by Millan and Manes (Proc. Natl. Acad. Sci. USA, 85: 3024-3028, 1988). Furthermore, the 3'-untranslated region of the LS174T cell alkaline phosphatase message is very similar to the sequence immediately downstream of the coding region of the germ cell alkaline phosphatase genomic DNA clone. Thus, these results indicate that this colon cancer cell alkaline phosphatase is likely to represent an allelic variant encoded at the germ cell alkaline phosphatase locus.

Alkaline Phosphatase↗
PubMed1975

Neutrophil alkaline phosphatase: comparison of enzymes from normal subjects and patients with polycythemia vera and chronic myelogenous leukemia.

To determine whether decreased alkaline phosphatase activity in the granules from neutrophils of patients with chronic myelogenous leukemia (CML) was due to an absence of enzyme or the production of defective enzyme, we compared the immunologic properties of granule alkaline phosphatase derived from patients with CML with that of normal subjects and patients with polycythemia vera (PRV). Antisera prepared in rabbits against granule alkaline phosphatase purified from the neutrophils of a patient with PRV produced a single precipitin line of antigenic identity when reacted with extracts of normal, PRV, and CML neutrophil granules. A histochemical stain for alkaline phosphatase activity (alpha-naphthyl acid phosphate coupled with Fast Blue RR) specifically stained the precipitin line. A variety of quantitative precipitin techniques failed to produce satisfactory precipitation of alkaline phosphatase activity. Comparative analyses were therefore performed by affinity chromatography using goat antirabbit-gammaglobulin linked to Sepharose 4B to adsorb alkaline phosphatase complexed with rabbit gamma globulin. With this method, 100% of CML, normal, and PRV alkaline phosphatase could be adsorbed. Using limiting concentrations of antibody, a proportionally smaller fraction of enzyme activity was absorbed as the concentration of PRV alkaline phosphatase or normal alkaline phosphatase was increased. Extracts of CML granules containing comparable amounts of protein but 200-fold less alkaline phosphatase activity per milligram did not specifically reduce adsorption. Thus, in CML, we found no evidence that the granulocytes contained a large amount of antigenically normal but enzymatically defective alkaline phosphatase. Examination of electron micrographs revealed no significant differences in the number or distribution of granules in the granulocytes of normal subjects or patients with PRV or CML. This suggests that the low level of neutrophil alkaline phosphatase in CML granulocytes is the result of decreased enzyme content and not a consequence of synthesis of catalytically defective enzyme.

Alkaline Phosphatase↗
PubMed1984

Induction of placental alkaline phosphatase biosynthesis by sodium butyrate.

Placental alkaline phosphatase activity was induced in choriocarcinoma cells by sodium butyrate. Butyrate stimulated de novo synthesis of the enzyme and the increase in phosphatase activity could be completely accounted for by the increase in phosphatase protein: the increases in placental alkaline phosphatase immunoactivity and placental alkaline phosphatase biosynthesis as measured by incorporation of the radioactive precursors, L-[35S]methionine, [3H]mannose, and [3H] glucosamine were similar to the increase in phosphatase activity. Sodium butyrate increased the rates of placental alkaline phosphatase biosynthesis but had no effect on the rate of placental alkaline phosphatase degradation or processing. Both control and butyrate-induced cells contained polypeptides of 61,500 and 64,500 apparent molecular weights that were identified as the precursor and fully processed forms of the placental alkaline phosphatase monomer, respectively. Further, processing of the 61,500-dalton polypeptide to the 64,500-dalton polypeptide involved the incorporation of additional glucosamine and N-acetylneuraminic acid moieties. Gel electrophoresis of anti-placental alkaline phosphatase-precipitable polypeptides from an in vitro protein-synthesizing system directed by RNA isolated from control or butyrate-induced cells demonstrated that sodium butyrate induced the synthesis of placental alkaline phosphatase mRNA. Our data indicate that sodium butyrate induces the specific transcription of the placental alkaline phosphatase gene.

Alkaline Phosphatase↗
PubMed1994

Concurrent purification of type-1 and type-2A protein phosphatase catalytic subunits.

We describe a simple purification scheme for the active catalytic subunits of both protein phosphatases type-1 and type-2A. The advantage of this procedure over others is that it produces intact proteins with high yield and specific activity and is suitable for either kilograms of tissue or a dish of cells. Type-1 and type-2A phosphatases from rabbit skeletal muscle were resolved on polylysine-agarose and subsequently obtained in homogeneous form. The phosphatases demonstrated characteristic properties. The phosphatase-1 catalytic subunit was inhibited by inhibitor-2 and phosphoinhibitor-1 whereas phosphatase-2A was not. The phosphatase activities were stable for years at -20 degrees C when stored in the presence of Mg2+ and glycerol. Based on the predicted sequence of the carboxyl terminus of each phosphatase, antibodies specific for phosphatases-1 and -2A were produced in rabbits using synthetic peptides as immunogens. Immunoblots showed complete specificity of these antibodies for their respective phosphatases and confirmed that the purified phosphatases has intact carboxyl termini. The purified catalytic subunits and antibodies will be useful for examining the regulation and the physiological roles of these protein phosphatases in cellular physiology.

Animals↗
PubMed1998

Myosin light chain phosphatase: subunit composition, interactions and regulation.

This review has presented some of the recent data on myosin phosphatase from smooth muscle. Although it is not conclusive, it is likely that most of the myosin phosphatase activity is represented by a holoenzyme composed of three subunits. These are: a catalytic subunit of 38 kDa of the type 1 phosphatase, probably the delta isoform (i.e. PP1c delta); a subunit of about 20 kDa whose function is not established; and a larger subunit that is thought to act as a target subunit. This is termed the myosin phosphatase target subunit, MYPT. Various isoforms of MYPT exist and the relatively minor distinctions are in the C-terminal leucine zipper motifs and/or with inserts in the central region. Many regions of the molecule are highly conserved, including the ankyrin repeats in the N-terminal part of the molecule and the sequence around the phosphorylation site. In addition, these isoforms all contain the four residue PP1c-binding motif (Arg/Lys-Val/Ile-Xaa-Phe). MYPT has been detected in a variety of cells and thus is not unique to smooth muscle. With phosphorylated myosin as substrate, the phosphatase activity of PP1c is low and is enhanced on addition of MYPT. It is assumed that MYPT functions as a target subunit and binds to both PP1c and substrate. The N-terminal fragment of MYPT is responsible for the activation of PP1c activity, but how much of the N-terminal sequence is required is not established. An important point is that activation is not a general effect and is specific for myosin. It is not known if other substrates may be targeted to MYPT. There are two binding sites for PP1c on MYPT: a strong site in the N-terminal segment (containing the 4-residue motif) and a weaker site in the ankyrin repeats, possibly in repeats 5, 6 and 7. The location(s) of the myosin-binding sites on MYPT is controversial, and binding of myosin, or light chain, to both N- and C-terminal fragments has been reported. Regulation of myosin phosphatase activity involves changes in subunit interactions, although molecular mechanisms are not defined. There are basically two theories proposed for phosphatase inhibition (i.e. as seen in the agonist-induced increase in Ca2+ sensitivity). One hypothesis is that phosphorylation of Myosin light chain phosphatase MYPT (at residue 654 or 695 of the gizzard MYPT isoforms or an equivalent residue) inhibits the activity of the MP holoenzyme. The kinase involved is not established, but may be an unidentified endogenous kinase or a RhoA-activated kinase. The latter is an attractive possibility because there is convincing evidence that RhoA plays a crucial role in the Ca(2+)-sensitizing process in smooth muscle. A second idea involves arachidonic acid. This is released via phospholipase A2 and could either interact directly with MYPT and cause dissociation of the holoenzyme (thus effectively reducing the phosphatase activity to that of the isolated catalytic subunit), or it could activate a kinase that would phosphorylate MYPT and inhibit the phosphatase. It is possible that MP activity may also be activated, for example, following increases in cAMP and/or cGMP. Evidence in support of this is very limited and under in vivo conditions the phosphorylation of MYPT by the respective kinases has not been demonstrated. There is, however, a tentative hypothesis based on in vitro data that phosphorylation of MYPT by PKA alters its cellular localization. This involves a shuttle between the dephosphorylated membrane-bound and inhibited state (at least towards P-myosin) to a phosphorylated cytosolic or cytoskeletal, and active state. The pathway(s) discussed above originates at the cell membrane and is carried via one or more messengers to the level of the contractile apparatus where it is manifested by regulation of phosphatase activity. Various components of the route have been identified, including RhoA and the atypical PKC isoforms, but more remain to be discovered. It is possible that more than one pathway, or cascade, is

Animals↗
PubMed1990

Activity of protein phosphatases against initiation factor-2 and elongation factor-2.

The protein phosphatases active against phosphorylase a, elongation factor-2 (EF-2) and the alpha-subunit of initiation factor-2 (eIF-2) [eIF-2(alpha P)] were studied in extracts of rabbit reticulocytes. Swiss-mouse 3T3 fibroblasts and rat hepatocytes, by use of the specific phosphatase inhibitors okadaic acid and inhibitor proteins-1 and -2. In all three extracts tested, both phosphatase-1 and phosphatase-2A contributed to overall phosphatase activity against phosphorylase and eIF-2(alpha P), but phosphatase-2B and -2C did not. In contrast, only protein phosphatase-2A was active against EF-2. Furthermore, in hepatocytes there was substantial type-2C phosphatase activity against EF-2, but not against phosphorylase or eIF-2 alpha. These findings in cell extracts were borne out by data obtained by studying the activities of purified protein phosphatase-1 and -2A against eIF-2(alpha P) and eIF-2(alpha P) was a moderately good substrate for both enzymes (relative to phosphorylase a). In contrast, EF-2 was a very poor substrate for protein phosphatase-1, but was dephosphorylated faster than phosphorylase a by protein phosphatase-2A. The implications of these findings for the control of translation and their relationships to previous work are discussed.

Animals↗
PubMed1963

AN ANALYSIS OF CELLULAR AND SUBCELLULAR SYSTEMS WHICH TRANSFORM THE SPECIES CHARACTER OF ACID PHOSPHATASE IN ACETABULARIA.

Several species-specific molecular forms of acid phosphatase are known to exist in the unicellular green alga Acetabularia. In graft combinations between cells of Acetabularia mediterranea (med) and Acicularia Schenckii (acic) the expression of the med phosphatase is dominant over acic phosphatase. There is good evidence that in such grafts the preexisting acic phosphatase is converted on the molecular level via an intermediate form to the med phosphatase. This conversion can be initiated by the transplantation of a med cell nucleus to an anucleate acic cell, but will also take place in grafts between anucleate med and anucleate acic cells, indicating that the direct participation of a cell nucleus is not required. An incomplete conversion of acic phosphatase, which terminates at the intermediate stage, is induced in acic cells by injection of a concentrated homogenate of med cytoplasm. A similar partial conversion occurs in vitro in a mixture of homogenates from med and acic cells. Subcellular particles, such as chloroplasts or mitochondria, can be removed from the homogenates by centrifugation without impairing the reactions leading to the intermediate phosphatase type. Experimental evidence suggests that the transformation of phosphatase types is enzymatically catalyzed and may involve the conjugation of small molecules with the phosphatase protein. It was shown, however, that sialic acid is not involved, since the incubation of med or acic homogenates with neuraminidase did not modify the electrophoretic mobility of either enzyme type. Another type of phosphatase, which occurs in Acetabularia erenulata (cren) and can be distinguished electrophoretically from the aforementioned types, is not subject to interaction. In various mono- and multi-nucleate graft combinations between cren cells on one hand, and med or acic cells on the other hand, the cren phosphatase is synthesized independently of the enzyme of the graft partner.

Acetabularia↗
PubMed1984

The protein phosphatases involved in cellular regulation. Glycolysis, gluconeogenesis and aromatic amino acid breakdown in rat liver.

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-bisphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phosphofructo-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation.

Allosteric Regulation↗
PubMed2000

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells.

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.

Animals↗
PubMed1990

Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity.

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.

Amino Acid Sequence↗
PubMed1988

Acute regulation of hepatic protein phosphatases by glucagon, insulin, and glucose.

The intravenous administration of glucagon to anesthetized rats resulted within 5 min in a 20% drop in the hepatic phosphorylase phosphatase activity, as measured in a post-mitochondrial supernatant at low dilution, but it did not affect the activity of glycogensynthase phosphatase. On the other hand, the injection of insulin plus glucose caused increases by about 35% in both phosphatase activities. Upon subcellular fractionation these effects were recovered in the cytosol, but not in the glycogen/microsomal fraction. However, activity changes in the latter fraction were observed after recombination with the liver cytosol from a hormone-treated animal. Preincubation of the liver cytosol with modulator protein (a specific inhibitor of type-1 protein phosphatases) cancelled the activity changes induced by insulin plus glucose. No hormonal effects on hepatic protein phosphatase activities were observed when the fractions were either diluted an additional 10-fold or pretreated with trypsin. An acute hormonal regulation of protein phosphatases could also be demonstrated in the perfused liver. When added to the perfusion medium, glucose as well as insulin increased the cytosolic protein phosphatase activities by about 25%. Their effect was additive, irrespective of the order of addition. On the other hand, the addition of glucagon and/or vasopressin resulted in a 20% drop in the phosphorylase phosphatase activity. The presence of glucagon did not interfere with the effectiveness of insulin, and vice versa. The changes in the phosphorylase phosphatase activities induced by glucagon, insulin, and glucose represented changes in the Vmax only. We propose that the acute control of the hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities is mediated by transferable, cytosolic effector(s).

Animals↗
PubMed1986

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases.

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.

Adenosine Triphosphate↗
PubMed1985

Rabbit liver phosphorylase a phosphatase: regulation by glucose and caffeine.

The modulation of three molecular forms of liver phosphorylase a phosphatases, the "catalytic" subunit phosphatase (35 000 relative mass (Mr) and phosphatases 2A1 and 2A2 by glucose and caffeine and some physiologically important compounds were studied, using 32P-labelled phosphorylase a obtained from rabbit liver as substrate. Glucose and caffeine showed independent and additive activations. The caffeine effect was seen at micromolar to millimolar concentrations and glucose caused activation even at concentrations below the normal blood glucose level. The nucleotides ATP and AMP, at their presumed physiological concentrations in the liver, were strongly inhibitory. Inhibition by these nucleotides and other inhibitors tested showed varied responses to the presence of the activators glucose and caffeine, depending on the phosphatase form. Thus, significant relief of ATP inhibition was afforded by glucose and caffeine acting independently for the 35 000 Mr phosphatase, whereas relief of inhibition for phosphatases 2A1 and 2A2 required a combination of glucose, caffeine, and Mg2+. The Km of the liver 35 000 Mr phosphatase was about 50 microM for the liver substrate as compared with 4 microM for the muscle substrate. The Km of phosphatase 2A2 was about 16 microM and for phosphatase 2A1 it was about 20 microM, using liver substrate in the absence of any stimulators. Mg2+ inhibited the 35 000 Mr phosphatase, but became stimulatory for phosphatase 2A2 and was an almost obligatory requirement for phosphatase 2A1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1967

[Studies On Phosphatase Activity In Some Parasitic Helminths]

In order to obtain some informations on the nature and relative activity of the phosphatases present in various helminths, biochemical studies have been made in thirteen kinds of worm parasites including the adults and larvae (Fasciola hepatica, Eurytrema pancreaticum, Paramphistomum sp., Taenia solium, Taenia pisiformis, Dipylidium caninum, Diphyllobothrium mansoni, Cysticercus cellulosae, Cysticercus fasciolaris and Sparganum). A comparison based on the analysis of pH-activity curves was made among these helminths. The worm materials were mostly obtained alive from an abattoir and removed from the organs or tissues of the animal hosts naturally infected. Sparganum and Cysticercus cellulosae, however, are collected from the subcutaneous tissue of the patients by surgical removal. The worms thoroughly washed were weighed and transferred with 0.1 M Tris buffer to a chilled glass grinder (Capacity; 15 ml) and homogenized in the cold. The homogenate was centrifuged at 5000 RPM for 30 minutes. The supernatant was pipetted off for determination of the phosphatase activity. Incubation mixtures consisted of 1 ml substrate, 1 ml buffer and 0.5ml extract. The buffers used were Tris (Hydroxymethyl) aminomethane and citric acid monohydrate and the substrate was paranitrophenyl phosphate (1 gm/25 ml). These mixtures were incubated at the temperature of 37 degrees C for 30 minutes in water bath. The absorbance or transferance of mixture was determined colorimetrically by "Spectronic 20 "spectrophotometer at 410 nm against a distilled water blank. The amount of phenol liberated was then calculated from a standard curve using phenol solutions. Controls consisted of unincubated mixtures. The results were deducted from this experiment. The phosphatase activity occurred over all parasitic helminths used in this experiment. In trematodes, pH-activity curves have demonstrated two peaks of phosphatase activity in Fasciola hepatica and Paramphistomum species. However the acid phosphatase activity was predominantly found and the alkaline phosphatase activity was found distinctly to be low in all three species. In Eurytrema pancreaticum, the pH-activity curves displayed two peaks in acid phosphatase activity, one at pH 5.0 and the other pH 9.0. In cestodes, both alkaline and acid phosphatase activity displayed the pH optima 5.0 and 9.0 to 10.0 in the adult tapeworms. However, major activity in the adults is due to the alkaline phosphtases. In contrast to the adults, Cysticercus and sparganum showed the higher activity in acid phosphatases which predominates in the larvae. In all cases of nematodes, the pH optimum for acid phosphatase was 4.0 to 6.0. A preponderance of acid phosphatase activity was shown in the extract of intestine of Ascaris lumbricoides. The aspect that phosphatases are correlated with phosphorylated passage of substances through the cuticle of helminths and may also be involved in carbohydrate metabolism is discussed.

Journal Article↗
PubMed1990

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle.

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.

Acid Phosphatase↗
PubMed1985

Immunological characterization of phosphoprotein phosphatases.

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.

Animals↗
PubMed1987

Purification and characterization of two phosphorylase phosphatases from rabbit liver.

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.

Adenosine Triphosphate↗