Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.
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PMID:Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding. 0 91

beta-Casein, and the phosphate containing peptide derived from it by tryptic digestion, have been dephosphorylated by the action of two phosphatases. Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to remove the phosphates from these substrates in two distinct stages. Substrate molecules retaining three of the original phosphoseryl residues accumulate during the reaction and are resistant to further dephosphorylation at low enzyme concentrations. In contrast bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) achieves complete dephosphorylation of these substrates sequentially without any of the intervening species showing resistance to the action of the enzyme. The phosphopeptide has been partially dephosphorylated by the action of the two phosphatases and the resultant peptides containing three phosphoseryl residues compared in their reactivity toward the E. coli alkaline phosphatase. The results obtained are discussed in relation to the mode of action of the two enzymes.
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PMID:A study of the enzymic dephosphorylation of beta-casein and a derived phosphopeptide. 18 32

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.
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PMID:Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation. 18 99

A metal-ion-independent, nonspecific phosphoprotein phosphatase (Mr = 35000) which represents the major phosphorylase phosphatase activity in bovine adrenal cortex has been purified to apparent homogeneity. An alkaline phosphatase activity (p-nitrophenyl phosphate as a substrate) of the same molecular weight, which requires both a metal ion (Mg2+ greater than Mn2+ greater than Co2+) and a sulfhydryl compound for activity, has been found to co-purify with the phosphoprotein phosphatase throughout the purification procedures. Characterization of the phosphoprotein and the alkaline phosphatase activities with respect to their catalytic properties, substrate and metal ion specificities, relationship with large molecular forms of the enzymes and responses to various effectors has been carried out. The results indicate that the phosphoprotein phosphatase can be converted by pyrophosphoryl compounds (e.g. PPi and ATP) to a metal-ion-dependent form which, subsequently, can be reactivated by Co2+ greater than Mn2+ but not by Mg2+ or Zn2+. The results also indicate that, although the phosphoprotein and the alkaline phosphatase activities are closely associated, they exhibit distinct physical and catalytic properties. Discussions concerning whether these two activities represent two different forms of the same protein or two different yet very similar polypeptide chains have been presented.
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PMID:Purification and properties of a phosphorylase (phosphoprotein) phosphatase associated with an alkaline phosphatase of Mr 35000 from bovine adrenal cortex. 23 Sep 63

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is reversibly inhibited by a variety of phenylarsonic acids, including some N-haloacetylated derivatives. The inhibition is of the competitive type, and Ki values are reported. The action on the enzyme of one of the arsonate inhibitors, the azo dye, 4-(4-aminophenylazo)-phenylarsonic acid was studied in detail, using spectrophotometric and kinetic methods. The azo dye binds more strongly to E. coli alkaline phosphatase than do the other arsonates. Spectrophotometric titration indicates the presence of a single, strong dye-binding site on the enzyme dimer molecule in the concentration range covered. In 0.1 M Tris - HCl buffer pH 8.0, 25 degrees C K diss for the dye - enzyme complex is 1.50 - 10(-5) M as determined by spectrophotometric titration. This value is in good agreement with the Ki = 1.30 - 10(-5) M obtained from kinetic measurements. The dye can be displaced from alkaline phosphatase by phosphate and competitive inhibitor 2-aminoethyl phosphonate. These results indicate that the dye binds with its arsonic acid group to the anion binding site of the active site of the enzyme. The binding of the dye to the native enzyme is associated with a red shift in the visible spectrum of the dye. It seems that the aromatic portion of the dye interacts with a hydrophobic region close to the anion binding site. The spectrum of the dye is not changed in the presence of the apoenzyme. When zinc is added to an apoenzyme-dye solution, the spectral changes of the dye depend on both the ratio of zinc per apoenzyme and the pH. The presence of Mg2+ had no effect on the observed phenomenon.
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PMID:Spectral studies of the interactions of Escherichia coli alkaline phosphatase with 4-(4-aminophenylazo)-phenylarsonic acid. 31 70

Cell-associated alkaline phosphatase (ALPase) of Bacteroides gingivalis 381 was found in the outer part of the periplasmic space by using an ultracytochemical procedure. Cell-associated ALPase was solubilized by extraction with 1% Triton X-100, and the solubilized enzyme was purified 904-fold with 5.6% recovery by using affinity column chromatography for mammalian intestinal-form ALPase. The purified enzyme gave a single protein band that corresponded to the enzyme activity band on polyacrylamide gel electrophoresis preparations. A single protein band at a molecular weight of 61,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis preparations. The molecular weight of the native enzyme was estimated to be 130,000 by gel filtration with TSK-gel G3000SW. These findings indicate that B. gingivalis ALPase is a homodimer. The optimal pH of the enzyme was between 9.1 and 9.3 in the absence of divalent metal ions and was between 10.1 and 10.3 in the presence of manganese or zinc ions. The apparent km for p-nitrophenylphosphate was 0.037 +/- 0.003 mM (mean +/- standard deviation) at pH 9.2 in the absence of divalent metal ions and 0.22 +/- 0.02 mM at pH 10.2 in the presence of 1 mM manganese ions. Under both of the conditions described above, the purified enzyme was able to hydrolyze casein and O-phosphoserine, suggesting that B. gingivalis ALPase can act as a phosphoprotein phosphatase. ALPase that immunologically cross-reacted with the purified enzyme was found in the extracellular soluble fraction. This means that ALPase is released from the periplasmic space into the culture supernatant as a soluble form.
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PMID:Purification and characterization of alkaline phosphatase of Bacteroides gingivalis 381. 211 73

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg2+ to rat liver 104,000 X g supernatant (S104) produced a 100-140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg2+. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg2+. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg2+ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg2+.
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PMID:Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C. 255 47

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.
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PMID:Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O. 264 90

The properties and developmental regulation of the protein phosphatases of Dictyostelium discoideum were examined. When crude extracts from vegetative cells were separated on a Mono Q column (FPLC) three protein phosphatase peaks, designated P1, P2 and P3 were found. When aggregation and culmination cells were examined only one protein phosphatase peak was observed. This corresponded to phosphatase P1 of vegetative cells. All three of the vegetative cell phosphatase were inhibited by heparin and mammalian phosphatase inhibitor-2, both of which are specific for type-1 protein phosphatases. Trifluoperazine, which inhibits type-2 protein phosphatases, had little effect on any peaks while levamisole, an alkaline phosphatase inhibitor, stimulated P2, slightly inhibited P3 and had no effect on P1. These results demonstrate the existence of two vegetative phase specific protein phosphatases in D. discoideum and one which occurs during all phases of the life cycle. The protein phosphatases isolated from vegetative cells all appear to be type-1 enzymes.
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PMID:Properties and developmental regulation of the protein phosphatases in Dictyostelium discoideum. 284 42

Cysteamine and propionitrile cause severe duodenal ulcers with perforation within 24-48 h after a single injection in rats. These animal models were used to gain insight into the early, preulcerogenic biochemical changes in the duodenal mucosa. The results indicate that a single sc injection of cysteamine and propionitrile induced dose- and time-dependent decreases in the activity of phosphoprotein phosphatase (PPPase) in homogenate and particulate fractions of rat duodenal mucosa. The decrease in enzyme activity was detectable 4 h after the injection of the ulcerogens, it was maximal at 12 h, and hardly detectable at 24 h. No effect on the enzyme activity was found under in vitro conditions. PPPase activity in the liver was not influenced by either cysteamine or propionitrile. Furthermore, the toxic but nonulcerogenic derivative of cysteamine ethanolamine had no effect on PPPase in the duodenum. Thus, the effect of the duodenal ulcerogens on PPPase activity was indirect and organ specific, related only to the target organ (i.e., duodenal mucosa). The effect of the drugs was also selective at the level of mucosal cells: both duodenal ulcerogens depleted protein and alkaline phosphatase but not lysosomal acid phosphatase. The decrease of PPPase activity could be a general property of the duodenal ulcerogens since it is independent of their effect on endogenous somatostatin.
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PMID:The influence of cysteamine and propionitrile on duodenal phosphoprotein phosphatase in rats. 289 91


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