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)

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of tri- and diphosphonucleosides and it requires either Ca2+ or Mg2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na+/K(+)-ATPase, to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap5A (100 microM), an inhibitor of adenylate kinase. In contrast, sodium azide (10 mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10 microM) and 5'-p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and DP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent Km and Vmax, obtained with ATP as the substrate, were evaluated at 23 +/- 3 microM and 1.09 mumol Pi/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.
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PMID:Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta. 147 95

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.
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PMID:Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas. 624 96

Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.
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PMID:Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes. 773 91

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca(2+)-ATP and Ca(2+)-ADP as substrates. Ca(2+)-ATP and Ca(2+)-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca(2+)-ATPase and Ca(2+)-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.
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PMID:Characterization of an ATP diphosphohydrolase activity (APYRASE, EC 3.6.1.5) in rat blood platelets. 817 26

ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.
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PMID:Purification and properties of human placental ATP diphosphohydrolase. 852 70

In the present report we describe an ATP diphosphohydrolase (apyrase EC 3.6.1.5) in rat cardiac sarcolemma. It is Ca2+ dependent and is insensitive to ouabain, orthovanadate, N-ethylmaleimide (NEM), lanthanum, and oligomycin that are classical ATPase inhibitors. Sodium azide that is a mitochondrial inhibitor at low concentrations, did not affect the enzyme activity at 5.0 mM or below. In contrast, at high concentrations (> 10 mM) sodium azide inhibited the enzyme. Levamisole, a specific inhibitor of alkaline phosphatase and P1, P5-di(adenosine 5'-)pentaphosphate (Ap5A), a specific inhibitor of adenylate kinase did not inhibit the enzyme. Mercury chloride showed a parallel inhibition of the hydrolysis of both substrates of apyrase. Similar inhibition profiles are powerful evidence for a common catalytic site for the hydrolysis of both substrates. The enzyme has an optimum pH range of 7.5-8.0 and catalyzes the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. The apparent Km (Michaelis constant) and Vmax (maximal velocity) are 62.1 +/- 5.2 microM and 1255.7 +/- 178 micromol inorganic phosphate liberated/min/mg with ATP and 59.4 +/- 4.3 microM and 269.2 +/- 39 micromol inorganic phosphate liberated/min/mg with ADP. Enzyme markers indicated that this apyrase is associated with the plasma membrane. A deposition of lead phosphate granules on the outer surface of the sarcolemmal vesicles was observed by electron microscopy in the presence of either ATP or ADP as substrate. It is suggested that the ATP diphosphohydrolase could regulate the concentration of extracellular adenosine, and thus is important in the control of vascular tone and coronary flow.
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PMID:Characterization and localization of an ATP diphosphohydrolase activity (EC 3.6.1.5) in sarcolemmal membrane from rat heart. 914 25

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.
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PMID:Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis. 1140 67

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
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PMID:Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes. 1186 92

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis. To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.
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PMID:Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process. 1263 29

Nucleotides, e.g. ATP and ADP, are important signaling molecules, which elicit several biological responses. The degradation of nucleotides is catalyzed by a family of enzymes called NTPDases (nucleoside triphosphate diphosphohydrolases). The present study reports the enzymatic properties of a NTPDase (CD39, apyrase, ATP diphosphohydrolase) in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for ATP and ADP hydrolysis in a pH range of 7.5-8.0 in the presence of Ca(2+) (5 mM). The enzyme displayed a maximal activity for ATP and ADP hydrolysis at 37 degrees C. It was able to hydrolyze purine and pyrimidine nucleosides 5'-di and triphosphates, being insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (0.1 mM). A significant inhibition of ATP and ADP hydrolysis (68% and 34%, respectively) was observed in the presence of 20 mM sodium azide, used as a possible inhibitor of ATP diphosphohydrolase. Levamisole (1 mM) and tetramisole (1 mM), specific inhibitors of alkaline phosphatase and P1, P(5)-di (adenosine 5'-) pentaphosphate, an inhibitor of adenylate kinase did not alter the enzyme activity. The presence of a NTPDase in brain membranes of zebrafish may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in central nervous system of this specie.
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PMID:ATP and ADP hydrolysis in brain membranes of zebrafish (Danio rerio). 1289 30


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