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)

We investigated factors influencing alkaline phosphatase activity in the course of developing criteria for the establishment of a standardized method for its determination in human serum at 30 degrees C. The effects of pH, phosphorylatable acceptor (2-amino-2-methyl-1-propanol and diethanolamine), 4-nitrophenyl phosphate, magnesium ion, zinc ion, temperature, volume fraction of specimen, and details of initiation of the reaction have been studied, with use of partly purified enzymes from bone, intestine, liver, and placenta, and sera from patients with a predominant characterized isoenzyme. The purity of the diethanolamine was examined and contaminant monoethanolamine was characterized as a competitive inhibitor. Two sets of recommended conditions are: 2-amino-2-methyl-1-propanol, 0.9 mol/liter; 4-nitrophenyl phosphate, 16 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/30, and pH30 degrees C 10.5; diethanolamine, 1.8 mol/liter; 4-nitrophenyl phosphate, 18 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/60, and pH30 degrees C 10.1. Serum is preincubated with all reagents but 4-nitrophenyl phosphate, which is used as the reaction-initiating substrate.
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PMID:Criteria for establishing a standardized method for determining alkaline phosphatase activity in human serum. 2 62

The stability of isoenzymes of alkaline phosphatase from liver, bones and small intestine was compared after addition to inactivated serum in the buffer systems: glycine, 2-amino-2-methyl-1-propanol, diethanolamine and 2-amino-2-methyl-1,3-propandiol at 37 degrees C. The mentioned isoenzymes were inactivated to different extents in glycine and 2-amino-2-methyl-1-propanol buffers. In diethanolamine and 2-amino-2-methyl-1,3-propandiol buffers sufficient stability of isoenzymes is obtained so that only these buffers are suitable for activity determinations of alkaline phosphatase at 37 degrees C.
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PMID:Stability of isoenzymes of alkaline phosphatase in various buffer systems. 43 6

The influence of Zn2+ on the inactivation of alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1 3.1] in serum during preincubation with 2-amino-2-methyl-1-propanol (AMP) buffers was investigated. Addition of Zn2+ to the buffer before preincubation increases the enzyme activity. An optimum Zn2+ concentration different for each lot of AMP buffer can be found, at which the enzyme activities are restored to a level equal to activities measured without preincubation. There is a relation between the inactivating properties of the different AMP buffers and the amount of Zn2+ needed to prevent this inactivation. Since Zn2+ chelating substituted diamines are held responsible for the inactivation by removing Zn2+ from the enzyme, we assume that the addition of Zn2+ to the buffer prevents this removal. As Zn2+ itself is an inhibitor of the enzyme, the addition of both too much or too little Zn2+ results in lower enzyme activities after preincubation with AMP buffer.
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PMID:Influence of zinc ions addition to different lots of 2-amino-2-methyl-1-propanol (AMP) buffer on the alkaline phosphatase activities. 49 32

Recent findings indicate that the buffer 2-amino-2-methyl-1-propanol, widely used in clinical measurement of alkaline phosphatase activity, contains an impurity that inhibits the activity of the enzyme. Analysis of several lots of 2-amino-2-methyl-1-propanol has demonstrated the presence of a material, the concentration of which correlates well with alkaline phosphatase activity. Mass spectral analysis of this material indicates that it is a substituted ethylenediamine compound. Zinc chelation is discussed as a possible mode of its inhibition. Further work is in progress to separate and characterize this and other compounds in the buffer and to determine their modes and degrees of alkaline phosphatase inhibition.
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PMID:An impurity in the buffer 2-amino-2-methyl-1-propanol, which correlates with depression of measured alkaline phosphatase activity. 68 26

Human serum incubated with 2-amino-2-methyl-1-propanol (860 mmol/liter) and magnesium ion (270 mumol/liter) at pH 10.35 showed greater alkaline phosphatase (EC 3.1.3.1) activity than if an equal amount of magnesium ion was added at the time of measurement. The apparent increase is due in part to a slight lability of serum alkaline phosphatase in 2-amino-2-methyl-1-propanol, which is prevented by the inclusion of magnesium. For some sera, however, a portion of this increased activity is real rather than artifactual. Use of serum rather than 4-nitrophenylphosphate to initiate the reaction produced relatively low activities and, in some cases, markedly nonlinear (increasing) rate progress curves. The behavior of some commercial lyophilized control sera differed significantly from that of patients' sera, in particular exhibiting a marked lability in the presence of 2-amino-2-methyl-1-propanol. Incubation of these labile materials with Mg2+ slightly improved their stability; addition of Zn2+ plus Mg2+ markedly stimulated and completely protected their alkaline phosphatase activity.
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PMID:Effect of incubation with Mg2+ on the measurement of alkaline phosphatase activity. 90 17

We evaluated N-methyl-D-glucamine (MEG) as a buffer for assay of alkaline phosphatase (ALP; EC 3.1.3.1) and compared the MEG-based assay with the current International Federation of Clinical Chemistry Reference Method for ALP (IFCC/RM/ALP), in which 2-amino-2-methyl-1-propanol (AMP) is the pH buffer. The ALP assay in MEG at 30 and 37 degrees C shows excellent correlation with the IFCC/RM/ALP at 30 degrees C, but yields proportionately higher ALP activities (8.2% at 30 degrees C and 57% at 37 degrees C). ALP is unstable in both MEG and AMP at 37 degrees C. Serum incubated in MEG undergoes a pH-dependent biphasic loss of ALP activity: an initial rapid 5% loss after 1 min of incubation and a 10% loss per hour thereafter. A similar pattern was seen for incubation with AMP. The use of a serum-initiated reaction (no preincubation of enzyme with buffer) eliminated the early loss in activity. The addition of the metal ion buffer N-hydroxyethylethylenediaminetriacetic acid, along with low concentrations of Zn and Mg, as used in the IFCC/RM/ALP, reduced the slow loss in activity over time, as did decreasing the reaction temperature to 30 degrees C, but had no effect on the early rapid decay in activity seen in the first minute. Moderate transphosphorylation (45%) and nonenzymatic hydrolysis (3.3 U/L) were observed with MEG under the conditions of the assay (37 degrees C). A comparison of different lots of MEG from two manufacturers showed no significant difference in ALP activities.
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PMID:Investigation of N-methyl-D-glucamine buffer for assay of alkaline phosphatase in serum. 142 26

Purified isoenzymes of human alkaline phosphatase from placenta, intestine and liver were investigated as catalysts for phosphotransferase activity, using the phosphoacceptors Tris, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, diethanolamine, 2-(ethylamino)ethanol, ethanolamine, and N-methyl-D-glucamine. All of the compounds supported phosphotransferase catalysis, conforming to saturation kinetics. There was little difference among the isoenzymes with respect to Km values of the acceptors, but the liver form was the most efficient (highest Vmax/Km) in forming phosphoacceptors; it was also the most efficient (highest Vamax/Ka) when the phosphoacceptors were considered as activators. At Vmax the isoenzymes differed little in their support of phosphotransferase activity relative to phosphohydrolysis, although the intestinal enzyme tended to be the poorest. The two best acceptors were diethanolamine, providing the highest phosphotransferase velocity, and 2-(ethylamino)ethanol, having the lowest Km. The phosphoaceptors that bound Zn2+ tightly did not function well in the phosphotransferase reaction, and vice versa. However, temporal assessment of the phosphohydrolytic and phosphotransferase activities during removal of Zn2+ from the enzyme with 1,10-phenanthroline revealed no evidence of a special role for Zn2+ in the latter activity.
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PMID:Phosphotransferase activity of human alkaline phosphatases and the role of enzyme Zn2+. 303 34

Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1.3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-1-propanol (AMP) buffer reagent system with 10 mmol/l levamisole (final assay concentration 8.1 mmol/l). The assay imprecision (SD) was 0.6 U/l compared with 3.9 U/l using L-phenylalanine for specimens with total alkaline phosphatase activity less than 250 U/l (reference range 30-120 U/l). In serum pools with raised total alkaline phosphatase activity errors in recovered intestinal activity were small (usually less than 3 U/l) when intestinal alkaline phosphatase was added. Much larger errors and many underestimated results were found using L-phenylalanine. For non-haemolysed specimens it is concluded that an assay based on levamisole inhibition provides a better measure of intestinal alkaline phosphatase activity than L-phenylalanine.
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PMID:Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component. 335 Sep 81

Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.
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PMID:Purification and partial characterization of alkaline phosphatase of matrix vesicles from fetal bovine epiphyseal cartilage. Purification by monoclonal antibody affinity chromatography. 396 87

We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.
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PMID:A reference method for measurement of alkaline phosphatase activity in human serum. 640 66


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