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)

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzyme antigens might be also interesting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg2+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-beta-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes.
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PMID:Parasite enzymes as a tool to investigate immune responses. 134 26

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed in Escherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42 degrees C. This enzyme required Zn2+ for its catalytic activity; however, Mg2+ or Ca2+ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn2+. Kinetics of Z. mobilis alkaline phosphatase production in E. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.
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PMID:Characterization of Zymomonas mobilis alkaline phosphatase activity in Escherichia coli. 136 89

Brush-border membranes from rat duodenal and jejunal mucosa were prepared by differential Ca(2+)-precipitation. Kinetical properties and Mg(2+)-stimulation of alkaline phosphatase were studied for the enzyme either bound to these membranes or purified from these membranes by liquid chromatography. With p-nitrophenylphosphate as substrate, the alkaline phosphatase apparent Km was lower in jejunum (90 microM) as compared with duodenum (160 microM), and lower for the purified enzyme (jejunum: 55 microM; duodenum: 97 microM) as compared to the bound one. In the presence of 5 mM MgCl2, the substrate affinity was in all cases decreased. For the bound enzyme Vmax was 10 times greater in duodenum compared to jejunum. 5 mM MgCl2 tripled the Vmax of the duodenal bound enzyme and increased it by 50% for the jejunal one, but a seven-fold increase was recorded for the purified enzyme at both levels of intestine. The apparent affinity for Mg2+ was similar for the bound and the free enzyme, for duodenum and for jejunum (Mg0.5: +/- 40 microM).
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PMID:Bound and purified alkaline phosphatase from rat duodenal and jejunal brush-border membranes: kinetics and magnesium stimulation. 138 Mar 28

1. Effect of intracellular ATP on Cl- current (ICl) mediated by the GABA (gamma-aminobutyric acid) receptor subtype, GABAA, was studied in dissociated nucleus tractus solitarii (NTS) neurones using the whole-cell mode of patch clamp. A concentration-jump technique termed 'Y tube' was used to rapidly apply agents externally. Dissociated neurones were obtained from 1- to 3-week-old rats. 2. When the patch-pipette solution contained 2 mM-ATP, the amplitude of ICl elicited by 10(-5) M-GABA did not show any time-dependent decrease (apparent run-down), for more than 60 min after the initial recording. In the presence of ATP, the half-maximum concentration (KD) and Hill coefficient calculated from the GABA concentration-response curve were 9.12 microM and 1.47, respectively. 3. In the absence of intracellular ATP, the amplitude of GABA-induced ICl decreased with time. The relative peak amplitudes after 20 and 60 min from the initial recording were 0.40 +/- 0.09 (n = 11) and 0.16 +/- 0.05 (n = 8) with respect to the initial response. 4. Removal of Mg2+ from the internal solution induced run-down of the GABA response even in the presence of 2 mM-intracellular ATP, suggesting that both intracellular ATP (2 mM or more) and Mg2+ are necessary to prevent run-down of the GABA response. 5. Activation of dephosphorylation processes by alkaline phosphatase (100-200 microM) did not affect the GABA response in neurones perfused with internal solution containing 2 mM-ATP and 3 mM-Mg2+. Blocking the dephosphorylation process by okadaic acid, a phosphatase inhibitor, did not prevent the run-down of the GABA response. 6. Calcium influxes passing through both the voltage-dependent L-type Ca2+ channel and the glutamate receptor-operated cation channel did not affect ICl induced by GABA. 7. GABA-induced ICl was also maintained by adding 2 mM-ADP or ATP gamma S (adenosine-5'-O-3-thiotriphosphate) to the internal solution containing Mg2+. Addition of 2 mM-adenosine, AMP, cyclic AMP, AMP-PNP (adenylimido-diphosphate) or ADP beta S (adenosine-5'-O-2-thiodiphosphate) to the internal solution did not prevent the run-down of the GABA response even in the presence of 3 mM-intracellular Mg2+. Based on the chemical specificity of these ATP analogues, it is suggested that there is an ATP-sensitive binding site (ATP receptor) in the cytoplasmic side of the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat. 138 52

The effects of divalent cations and of some inhibitors on the activities of alkaline phosphatase and ATPase were examined in rat jejunal brush-border membranes (BBM) isolated by tha Ca(2+)-(BBMCa) or the Mg(2+)-precipitation method (BBMMg). Similar results were found in BBMCa and BBMMg though generally higher in BBMCa. Alkaline phosphatase activity was stimulated by 5 mM MgCl2 (30% to 44%), but not by 5 mM CaCl2 or 0.1 mM ZnCl2, at pH 9.5 or 7.4. ATPase activity was equally stimulated by 5 mM MgCl2 and by 5 mM CaCI2 (about 150%). Alkaline phosphatase activity was significantly inhibited by 1 mM vanadate, 5 mM diamox, 5.0 mM L-leucine and 1 mM theophylline. In contrast, Ca(2+)-ATPase and Mg(2+)-ATPase activities were not depressed by those alkaline phosphatase inhibitors, but were inhibited by 0.1 mM trifluoperazine (more than 70%). 0.1 mM ZnCl2 also appeared to be inhibitory to Ca(2+)-ATPase and Mg(2+)-ATPase, but not to alkaline phosphatase activity even in the presence of Ca2+ and Mg2+. These results suggest that Ca(2+)-ATPase and Mg(2+)-ATPase activities of the rat jejunal BBM are not merely manifestations of alkaline phosphatase, but rather belong to (a) distinct enzyme(s).
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PMID:Alkaline phosphatase and ATPases in brush-border membranes of rat jejunum: distinct effects of divalent cations and of some inhibitors. 138 82

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.
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PMID:Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine. 145 47

It was found that mitochondria from human placenta exhibited an ADPase activity with the following characteristics. The enzyme responsible for this activity was associated with the inner mitochondrial membrane. It was not released by treatment of the submitochondrial particles with solutions of high ionic strength. Maximal ADP hydrolysis was reached at pH 8. Specific inhibitors for alkaline phosphatase (L-phenylalanine), myokinase (P1,P5-di(adenosine-5')pentaphosphate), or 5'-nucleotidase (concanavalin A) did not decrease ADP hydrolysis. ATP synthesis from ADP by myokinase was about 13 nmol/mg/min, whereas ADP hydrolysis reached values around 500 to 550 nmol/mg/min, indicating that a myokinase-H+ATPase combination could not account for the observed rates of ADP hydrolysis. The activity was stimulated by Mg2+, but high concentrations of this cation produced inhibition. High ADP concentrations did not inhibit ADPase activity. Kinetic measurements of the activity in the submitochondrial particles showed that the true substrate was ADP-Mg. The kinetic studies showed V(app) values of 476 and 270 nmol/mg/min, and Kmapp values of 416 and 8.7 microM.
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PMID:Subcellular localization and properties of adenosine diphosphatase in human placenta. 147 Jun 6

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

Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn. In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+. The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer. The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+. In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8). In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9. D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates. As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2). The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.
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PMID:Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase. 152 59

Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.
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PMID:Detection of point mutation in bla T genes of Enterobacteriaceae by biotinylated oligonucleotide probes using microwell hybridization and enzymofluorometric method. 154 33


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