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)

Alkaline phosphatase has been purified from microsomes of chicken epiphyseal cartilage by first selectively extracting certain adventitious proteins with 0.25 M trichloroacetate. The membrane-bound enzyme was then solubilized by 1% cholate in buffered 33% saturated ammonium sulfate and purified by column chromatography on Bio-Gel A-5m, extraction with 1-butanol, and ion exchange chromatography on DEAE-Bio-Gel A. The purified alkaline phosphatase from the cartilage membrane had a subunit molecular weight of 53,000 and a holoenzyme weight of 207,000-220,000, indicating a tetramer. The pH optima for p-nitrophenylphosphate, ATP, and pyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively. Values of Vmax (in micromoles/min/mg) were 220, 3.1, and 0.8, respectively. Substrate inhibition was pronounced at values of pH below 8.5. Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 showed that phosphate and arsenate were competitive inhibitors (KI = 1.88 and 0.15 mM, respectively) and levamisole was an uncompetitive inhibitor (KI = 0.32 mM), while L-phenylalanine and ZnCl2 were mixed inhibitors (KI = 15.8 and 0.02 mM, respectively). Inhibition by preincubation in 1 mM EDTA was reversible by readdition of 0.25 mM MgCl2 nd 20 microM ZnCl2. The data indicate that this membrane-bound alkaline phosphatase from chicken epiphyseal cartilage is a Zn2+ and possibly Mg2+-containing enzyme. While the subunit molecular weight and kinetic properties of the enzyme are quite typical of vertebrate alkaline phosphatases, the tightness of binding to the membrane lipids, the extreme sensitivity to substrate inhibition, and the tetrameric conformation of the holoenzyme are unusual.
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PMID:Purification and initial characterization of intrinsic membrane-bound alkaline phosphatase from chicken epiphyseal cartilage. 725 97

We describe a method for determining serum alkaline phosphatase activity with use of N-methyl-D-glucamine buffer, Na+ is a definite activator, whereas NH4+ and Li+ inhibit enzyme activity. Optimum reaction conditions are: methylglucamine buffer, 0.35 mol/L, pH 10.2 +/- 0.1 (30 degrees C); NaCl, 70 mmol/L; MgCl2, 0.5 mmol/L; disodium 4-nitrophenyl phosphate, 15 mmol/L; reaction temperature, 30 degrees C; reaction time, 2 min. The assay conditions are optimum for all human serum isoenzymes.
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PMID:Use of N-methyl-D-glucamine as buffer in the determination of serum alkaline phosphatase activity. 728 25

Soon after the reconstitution of the lyophylized commercial control sera the alkaline phosphatase activity is smaller than stated value and theoretical value is reached after a time varying according to the various sera; at any rate this value is hever higher than seven hours since reconstitution. The lyophylized sera reconstitution with a solution of diethanolamine 1 mol/l or MgCl2 0,5 mmol/l alone or associate consents to restore much more swiftly the enzymatic activity to a higher value than 90% of the stated value since reconstitution. No alkaline phosphatase variation is observed in the congealed human sera at --20 degrees C after the decongealment. We recommend to reconstitute the lyophylized sera for the alkaline phosphatase determination by the use of a solution of diethanolamine 1 mol/l and MgCl2 0,5 mmol/l instead of distilled water.
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PMID:[The alkaline phosphatase activity in the lyophylized commercial control sera after reconstitution]. 745 65

In rat jejunal brush-border membranes (BBM), ATP hydrolysis activity was specifically stimulated by CaCl2 and by MgCl2, allowing to identify Ca(2+)-ATPase and Mg(2+)-ATPase activities with a broad pH optimum near 8.0. Nonspecific ATPase activity (in the absence of cations) had a pH optimum above 9.5 as alkaline phosphatase. The effects of Ca2+ and Mg2+ concentrations on ATPase activity evidenced two apparent KA for each cation. At high concentrations, a similar affinity for both cations was recorded (KA: 0.35 mM). At low concentrations, the affinity for Mg2+ was greater than for Ca2+ (KA: 0.02 mM and 0.07 mM respectively). In an attempt to differentially solubilize alkaline phosphatase and ATPase activities, eleven different detergents were assayed. They more or less successfully released Ca(2+)-ATPase and Mg(2+)-ATPase activities from BBM but the more membranes were solubilized by a detergent, the more activities were lost, suggesting a close dependence on integration in BBM. As to alkaline phosphatase and nonspecific ATPase, they almost co-solubilized with Ca(2+)-ATPase and Mg(2+)-ATPase but their total activity was little affected. After treatment of BBM with phosphatidylinositol-specific phospholipase C (E.C. 3.1.4.10), 58% of alkaline phosphatase activity and 45% of nonspecific ATPase activity were released in the supernatant while Ca(2+)-ATPase and Mg(2+)-ATPase activities remained totally incorporated in BBM pellets. These last results definitively demonstrate that Ca(2+)-ATPase and Mg(2+)-ATPase activities are not manifestations of alkaline phosphatase, as earlier suggested, but rather result from the existence of one or several intrinsic membrane enzymes.
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PMID:Ca(2+)-ATPase and Mg(2+)-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes. 751 33

We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.
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PMID:Kinetic studies on the membrane form of intestinal alkaline phosphatase. 755 5

Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization.
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PMID:Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase. 763 51

The activity of 1-alkyl-sn-glycero-3-phosphate (AGP) acetyltransferase was studied using microsomal fractions isolated from cerebral cortices of 15-day-old rabbits. Fraction P3A was isolated using buffered 0.32 M sucrose containing mercaptoethanol, EDTA and NaF. This fraction had specific AGP acetyltransferase activities which were 4.9-times those of microsomal fraction P3B isolated in 0.32 M sucrose alone. This P3B activity was increased 2.4-times after a preincubation in the presence of ATP, MgCl2 and a high-speed supernatant fraction from cerebral cortex. Further, the activities of both P3A and P3B were almost completely eliminated by preincubation in the presence of alkaline phosphatase. Thus an activation of the AGP acetyltransferase by phosphorylation was indicated. While there was little inhibition of the P3A AGP acetyltransferase in the presence of added ATP, the magnesium salt form of ATP (1 mM) was severely inhibitory, bringing about 86% inhibition for P3A and 91% for P3B. The inhibitory effects of MgADP and MgAMP were smaller, and MgATP was a much more effective inhibitor than MgCTP, MgGTP and MgUTP which brought about 20-38% inhibitions of P3A activity at 1 mM concentrations. The effect of MgATP may be of particular relevance to the synthesis of platelet activating factor (PAF) following a period of ischemia in brain. Falling MgATP levels during energy failure could relieve the inhibition of AGP acetyltransferase seen in healthy cells and allow the formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphate, which is the first committed intermediate in the de novo pathway of PAF synthesis.
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PMID:MgATP inhibits the synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphate by microsomal acetyltransferase of immature rabbit cerebral cortex. 801 76

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl2 and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca2+/calmodulin; this latter effect was blocked by the chelator ethylene-glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca2+, Ca2+/calmodulin, Mg2+ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca2+/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca2+/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.
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PMID:Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation. 813 99

In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased. Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.
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PMID:Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase. 864 34

Osteoporosis and magnesium (Mg) deficiency often occur in malabsorption syndromes such as gluten-sensitive enteropathy (GSE). Mg deficiency is known to impair parathyroid hormone (PTH) secretion and action in humans and will result in osteopenia and increased skeletal fragility in animal models. We hypothesize that Mg depletion may contribute to the osteoporosis associated with malabsorption. It was our objective to determine Mg status and bone mass in GSE patients who were clinically asymptomatic and on a stable gluten-free diet, as well as their response to Mg therapy. Twenty-three patients with biopsy-proven GSE on a gluten-free diet were assessed for Mg deficiency by determination of the serum Mg, red blood cell (RBC) and lymphocyte free Mg2+, and total lymphocyte Mg. Fourteen subjects completed a 3-month treatment period in which they were given 504-576 mg MgCl2 or Mg lactate daily. Serum PTH, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and osteocalcin were measured at baseline and monthly thereafter. Eight patients who had documented Mg depletion (RBC Mg2+ < 150 microM) underwent bone density measurements of the lumbar spine and proximal femur, and 5 of these patients were followed for 2 years on Mg therapy. The mean serum Mg, calcium, phosphorus and alkaline phosphatase concentrations were in the normal range. Most serum calcium values fell below mean normal and the baseline serum PTH was high normal or slightly elevated in 7 of the 14 subjects who completed the 3-month treatment period. No correlation with the serum calcium was noted, however. Mean serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and osteocalcin concentrations were also normal. Despite only 1 patient having hypomagnesemia, the RBC Mg2+ (153 +/- 6.2 microM; mean +/- SEM) and lymphocyte Mg2+ (182 +/- 5.5 microM) were significantly lower than normal (202 +/- 6.0 microM, p < 0.001, and 198 +/- 6.8 microM, p < 0.05, respectively). Bone densitometry revealed that 4 of 8 patients had osteoporosis of the lumbar spine and 5 of 8 had osteoporosis of the proximal femur (T-scores < or = -2.5). Mg therapy resulted in a significant rise in the mean serum PTH concentration from 44.6 +/- 3.6 pg/ml to 55.9 +/- 5.6 pg/ml (p < 0.05). In the 5 patients given Mg supplements for 2 years, a significant increased in bone mineral density was observed in the femoral neck and total proximal femur. This increase in bone mineral density correlated positively with a rise in RBC Mg2+. This study demonstrates that GSE patients have reduction in intracellular free Mg2+, despite being clinically asymptomatic on a gluten-free diet. Bone mass also appears to be reduced. Mg therapy resulted in a rise in PTH, suggesting that the intracellular Mg deficit was impairing PTH secretion in these patients. The increase in bone density in response to Mg therapy suggests that Mg depletion may be one factor contributing to osteoporosis in GSE.
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PMID:Magnesium deficiency: possible role in osteoporosis associated with gluten-sensitive enteropathy. 911 91


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