Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

1976 ◽  
Vol 22 (7) ◽  
pp. 972-976 ◽  
Author(s):  
H Van Belle
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Human Liver ◽  
Alkaline Phosphatases ◽  
Mechanism Of Inhibition ◽  
Optimum Ph ◽  
Alkaline Phosphatase Isoenzymes

Abstract I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

1990 ◽  
Vol 68 (9) ◽  
pp. 1112-1118 ◽  
Author(s):  
Lee Kihn ◽  
Dorothy Rutkowski ◽  
Robert A. Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Human Liver ◽  
Red Cell ◽  
Osteosarcoma Cells ◽  
Alkaline Phosphatases ◽  
Membrane Anchor ◽  
Gradient Gel Electrophoresis ◽  
Triton X ◽  
Phosphatidylinositol Phospholipase C

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 × slower in liposomes and 100 × slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.Key words: alkaline phosphatase, liposome, phosphatidylinositol, membrane anchor.

Alkaline phosphatase isoenzymes.

1982 ◽  
Vol 28 (10) ◽  
pp. 2007-2016 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Quantitative Analysis ◽  
Posttranslational Modifications ◽  
Quantitative Methods ◽  
Molecular Forms ◽  
Future Research ◽  
Alkaline Phosphatases ◽  
Isoenzyme Analysis ◽  
Alkaline Phosphatase Isoenzymes ◽  
Alkaline Phosphatase Isoenzyme

Abstract The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.

Use of specific inhibitors to disciminate alkaline phosphatase isoenzymes originating from human liver, placenta and intestine: absence of meconial alkaline phosphatase in maternal serum.

1979 ◽  
Vol 25 (7) ◽  
pp. 1230-1233 ◽  
Author(s):  
G J Doellgast ◽  
P J Meis
Keyword(s):  
Alkaline Phosphatase ◽  
Amniotic Fluid ◽  
Human Liver ◽  
In Utero ◽  
Maternal Serum ◽  
Alkaline Phosphatase Isoenzymes ◽  
High Concentrations ◽  
Specific Inhibitors

Abstract We used the inhibitors bromotetramisole, L-phenylalanine amide, and L-phenylalanine in combination to measure intestinal phosphatase in maternal serum and amniotic fluid. By using high concentrations of these inhibitors, it was possible to measure the three isoenzymes separately. We found no evidence of the presence of meconial alkaline phosphatase in the serum of the mother (six cases) after meconial passage in utero.

scholarly journals Isoelectric focusing of human liver alkaline phosphatase

1970 ◽  
Vol 118 (2) ◽  
pp. 299-302 ◽  
Author(s):  
A. L. Latner ◽  
Mary E. Parsons ◽  
A. W. Skillen
Keyword(s):  
Alkaline Phosphatase ◽  
Density Gradient ◽  
Isoelectric Focusing ◽  
Isoelectric Point ◽  
Human Liver ◽  
Sucrose Density Gradient ◽  
Ph Gradient ◽  
Alkaline Phosphatases ◽  
Carrier Ampholytes

1. Isoelectric focusing of human liver alkaline phosphatase in a sucrose density gradient with LKB Ampholine as carrier ampholytes is described. 2. Problems due to the chelating properties of the ampholytes and the pH gradient were examined. 3. A reactivation procedure to counter these effects was devised that can probably be used for other alkaline phosphatases. 4. The isoelectric point of human liver alkaline phosphatase was found to be pH3.9.

scholarly journals Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas

1981 ◽  
Vol 194 (3) ◽  
pp. 857-866 ◽  
Author(s):  
H Galski ◽  
S E Fridovich ◽  
D Weinstein ◽  
N De Groot ◽  
S Segal ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Time Course ◽  
De Novo ◽  
Placental Tissue ◽  
First Trimester ◽  
Alkaline Phosphatases ◽  
Heat Stable ◽  
Alkaline Phosphatase Isoenzymes ◽  
Specific Activities

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.

Biochemical and histochemical studies of alkaline and acid phosphatases in a digenetic trematode,Pegosomum egretti

1986 ◽  
Vol 60 (4) ◽  
pp. 293-298 ◽  
Author(s):  
Indra Rajvanshi ◽  
K. L. Mali
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Raw Materials ◽  
Digenetic Trematode ◽  
Acid Phosphatases ◽  
Alkaline Phosphatases ◽  
Optimum Ph ◽  
Standard Techniques ◽  
Alkaline And Acid Phosphatases

ABSTRACTThe biochemistry and histochemistry ofPegosomum egrettihave been studied using standard techniques. Phosphatases were analysed colorimetrically; the optimum pH for acid phosphatase activity was 5·0 and for alkaline phosphatase was 10·0. The results were compared with those of other trematodes. Histochemical localization of acid and alkaline phosphatases revealed differences in enzyme activity in various tissues. These differences in the site and pattern of distribution of the two enzymes have been discussed in relation to transport of raw materials and the metabolism of the cell concerned.

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

1987 ◽  
Vol 33 (5) ◽  
pp. 653-657 ◽  
Author(s):  
P Sorroche ◽  
A Bianchi-Bosisio ◽  
P K Sinha ◽  
C Gelfi ◽  
P G Righetti
Keyword(s):  
Alkaline Phosphatase ◽  
Isoelectric Focusing ◽  
Serum Alkaline Phosphatase ◽  
Normal Serum ◽  
Alkaline Phosphatases ◽  
Ph Gradients ◽  
Carrier Ampholytes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Serum Alkaline Phosphatase Isoenzymes ◽  
Acidic Components

Abstract This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

Separation of alkaline phosphatase isoenzymes from human kidney and comparison with alkaline phosphatases from other human tissues, urine, and Escherichia coli.

1983 ◽  
Vol 29 (1) ◽  
pp. 100-106 ◽  
Author(s):  
A W Hodson
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Human Kidney ◽  
Human Tissues ◽  
Ammonium Sulfate Precipitation ◽  
Alkaline Phosphatases ◽  
Alkaline Phosphatase Isoenzymes ◽  
Starch Gel

Abstract Human kidney isoenzymes of alkaline phosphatase (EC 3.1.3.1) after extraction with butan-1-ol were separated by ammonium sulfate precipitation, gel filtration, and chromatofocusing fractionation methods. The separation at each fractionation step was monitored by starch gel and equilibrium-gradient-pore electrophoresis, the latter technique also being used to determine molecular mass. The determined molecular mass (daltons) of alkaline phosphatase from human placenta was 132 000, from urine 95 000, and three isoenzymes from kidney were 195 000, 140 000, and 95 000, respectively. The mass of Escherichia coli alkaline phosphatase was 80 000 daltons, and that of human liver alkaline phosphatase was assumed to be 160 000 daltons. The urinary isoenzyme and the electrophoretically fastest migrating kidney isoenzyme were similar with regard to pH optima, charge, and molecular mass as well as response to L-phenylalanine, L-homoarginine, heat, and urea. Bacterial alkaline phosphatase could be distinguished from the alkaline phosphatases in human tissues and urine by differences in the response to changes in pH and several other physicochemical properties.

scholarly journals The action of cyanate on human and pig kidney alkaline phosphatases

1969 ◽  
Vol 111 (5) ◽  
pp. 745-748 ◽  
Author(s):  
M. J. Carey ◽  
P. J. Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Ph Dependence ◽  
Thiol Group ◽  
Prolonged Treatment ◽  
Enzyme Molecule ◽  
Amino Groups ◽  
Alkaline Phosphatases ◽  
Pig Kidney ◽  
Substrate Concentrations

1. At concentrations of cyanate up to 0·2m there is an apparently reversible combination with alkaline phosphatase (EC 3.1.3.1), but higher concentrations inhibit alkaline phosphatase irreversibly by a process that is time-dependent. 2. The effect of 0·2m-cyanate on the enzymic reaction velocity depends on the substrate concentration. There is inhibition when the substrate concentration is 1·0mm or higher, but at lower substrate concentrations cyanate has an activating effect. 3. The pH-dependence of the reversible reaction suggests that cyanate may react with a thiol group at or near the active site of the enzyme, preventing a conformational change that is believed to be important in the mechanism of action of alkaline phosphatase. 4. Prolonged treatment with 0·6m-cyanate probably carbamoylates all free amino groups in the enzyme molecule and generates a new enzyme with decreased Vmax. and increased Km.

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