scholarly journals The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1969 ◽  
Vol 112 (5) ◽  
pp. 699-701 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Competitive Inhibitor ◽  
Inorganic Pyrophosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Low Concentrations ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of ◽  
Pyrophosphate Hydrolysis

1. The differential effects of adding Zn2+ and Mg2+ on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn2+, inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn2+–pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg2+ also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.

scholarly journals Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1967 ◽  
Vol 102 (3) ◽  
pp. 917-921 ◽  
Author(s):  
R.H. Eaton ◽  
D. W. Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Competitive Inhibitor ◽  
Tissue Preparation ◽  
Intestinal Alkaline Phosphatase ◽  
Active Centre ◽  
Low Concentrations ◽  
Type 3 ◽  
Intestinal Preparation ◽  
Hydrolysis Of ◽  
Single Enzyme

1. Inhibition of the pyrophosphatase and orthophosphatase activities of human liver and small-intestinal alkaline-phosphatase preparations by different classes of inhibitors has been studied. 2. Each type of substrate, pyrophosphate or orthophosphate, is a competitive inhibitor of hydrolysis of the other type. 3. l-Phenylalanine is a non-competitive inhibitor of both types of activity of the intestinal preparation, but inhibits neither activity of the liver enzyme. Arsenate is a competitive inhibitor of both activities of both preparations. For a given inhibitor, the values of K(i) are independent of the type of substrate used when measurements are made at the same pH. 4. Mg(2+) ions activate orthophosphatase but inhibit pyrophosphatase, except in very low concentrations. 5. These results are compatible with the presence in each tissue preparation of a single enzyme with one type of active centre, possessing both orthophosphatase and pyrophosphatase activities.

An Immunoprecipitation Assay for High Molecular Weight Alkaline Phosphatase in Human Serum

1989 ◽  
Vol 26 (2) ◽  
pp. 151-157 ◽  
Author(s):  
Gerald A Maguire ◽  
Halima Adnan
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Liver Disease ◽  
High Molecular Weight ◽  
Solid Phase ◽  
Bone Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Molecular Weight Form ◽  
Hepatic Malignancies

The serum of patients with obstructive liver disease may contain a high molecular weight form of alkaline phosphatase (high Mr alkaline phosphatase). The presence of this form of alkaline phosphatase is associated with hepatic malignancies. We have investigated the use of anti-alkaline phosphatase monoclonal antibodies which do not bind high Mr alkaline phosphatase in assays for high Mr alkaline phosphatase. Direct immunoprecipitation of liver and bone alkaline phosphatase with solid phase anti-liver alkaline phosphatase antibody (which also reacts with bone alkaline phosphatase) and measurement of the residual supernatant alkaline phosphatase activity led to a precise assay. Intestinal alkaline phosphatase interfered in this assay which, consequently, was of little use in the differential diagnosis of liver disease. Indirect precipitation of liver, bone, placental and intestinal alkaline phosphatase by soluble anti-liver alkaline phosphatase (which reacts with liver and bone alkaline phosphatases), soluble anti-intestinal alkaline phosphatase (which reacts with placental and intestinal alkaline phosphatases) and solid phase anti-mouse IgG led to an assay which, although less precise, showed more promise of being useful clinically.

scholarly journals Chicken Intestinal Alkaline Phosphatase

1960 ◽  
Vol 43 (6) ◽  
pp. 1149-1169 ◽  
Author(s):  
M. Kunitz
Keyword(s):  
Alkaline Phosphatase ◽  
Zinc Ions ◽  
Magnesium Ions ◽  
Intestinal Alkaline Phosphatase ◽  
Reversible Inactivation ◽  
Higher Temperature ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Zn Ions ◽  
Denaturation Of Proteins

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.

Inorganic Pyrophosphatase Activity of Purified Bovine Pulp Alkaline Phosphatase at Physiological pH

1986 ◽  
Vol 65 (2) ◽  
pp. 125-127 ◽  
Author(s):  
M. Harada ◽  
N. Udagawa ◽  
K. Fukasawa ◽  
B.Y. Hiraoka ◽  
M. Mogi
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Pyrophosphatase ◽  
Pyrophosphatase Activity

Anion-stimulated ATPase activity of brush border from rat small intestine.

1979 ◽  
Vol 236 (1) ◽  
pp. E70 ◽  
Author(s):  
M H Humphreys ◽  
L Y Chou
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Brush Border ◽  
Atp Hydrolysis ◽  
Intestinal Transport ◽  
Mitochondrial Enzymes ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Small Intestinal

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.

THE METABOLISM OF THE ERYTHROCYTE: X. THE INORGANIC PYROPHOSPHATASE OF THE ERYTHROCYTE

1956 ◽  
Vol 34 (1) ◽  
pp. 121-129 ◽  
Author(s):  
A. Malkin ◽  
O. F. Denstedt
Keyword(s):  
Enzyme Activity ◽  
Calcium Ions ◽  
Human Erythrocyte ◽  
Inorganic Pyrophosphatase ◽  
Magnesium Ions ◽  
Constant Ratio ◽  
Inorganic Pyrophosphate ◽  
Noncompetitive Inhibitor ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of

The activity of the pyrophosphatase which catalyzes the hydrolysis of inorganic pyrophosphate in the erythrocyte of the human, the rabbit, and the chicken is confined entirely to the cytoplasm of the cell. Following preincubation, the enzyme activity in the human erythrocyte is diminished, but pre-incubation in the presence of cysteine or glutathione prevents the diminution of the enzyme activity. Aging of the hemolyzate of the human erythrocytes results in a marked loss of the inorganic pyrophosphatase activity. The diminished activity can be restored by the addition of cysteine or glutathione to the reaction mixture; but after the hemolyzate has aged for five or six days at 5 °C, the loss in the enzyme activity can no longer be restored with these reagents. Fluoride and calcium ions inhibit the activity of the enzyme, while magnesium ions are essential for its activity. Calcium is a noncompetitive inhibitor, while the inhibition by fluoride is of a "quadratic" nature. If a constant ratio of magnesium to pyrophosphate is maintained, the quadratic inhibition can be converted to the "uncompetitive" type of inhibition.

A fetal intestinal-type alkaline phosphatase in hepatocellular carcinoma tissue.

1977 ◽  
Vol 23 (9) ◽  
pp. 1615-1623 ◽  
Author(s):  
K Higashino ◽  
R Otani ◽  
S Kudo ◽  
Y Yamamura
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Mobility ◽  
Normal Liver ◽  
Heat Stability ◽  
Intestinal Type ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Adult Type ◽  
Alkaline Phosphatase Isoenzyme ◽  
Hepatocellular Carcinoma Tissue

Abstract We examined 19 hepatoma tissues for alkaline phosphatase isoenzyme and found that six have both the Kasahara isoenzyme and an alkaline phosphatase with a unique electrophoretic mobility, in addition to the liver-type enzyme. From two of six carcinoma tissues, the abnormal enzyme was partly purified and subjected to a detailed analysis, which clarified that the abnormal enzyme resembled a fetal intestinal alkaline phosphatase in most of its enzymic and immunologic properties and also in properties that reflect enzyme structure. This fetal intestinal-type alkaline phosphatase was not found in 24 specimens of normal liver from adults. The relevance of fetal intestinal-type alkaline phosphatase to Kasahara isoenzyme and adult intestinal alkaline phosphatase is discussed. The fetal and adult intestinal alkaline phosphatases differ in electrophoretic mobility, heat stability, and reactivity with concanavalin A. The adult-type enzyme has two components; only the electrophoretically slower, neuraminidase-resistant one is described here.

THE ROLE OF ALKALINE PHOSPHATASE IN INTESTINAL ABSORPTION I. THE KINETICS OF PHOSPHATASE ACTION ON VARIOUS SUBSTRATES

1953 ◽  
Vol 31 (1) ◽  
pp. 1-7
Author(s):  
Neil B. Madsen ◽  
Jules Tuba
Keyword(s):  
Alkaline Phosphatase ◽  
Average Energy ◽  
Inorganic Phosphorus ◽  
Intestinal Alkaline Phosphatase ◽  
Egg Lecithin ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of intestinal alkaline phosphatase action on sodium β-glycerophosphate, glucose 6-phosphate, and egg lecithin have been studied and compared. The Michaelis constants indicate that the enzyme shows considerably less affinity for lecithin than for the other two substrates, and the approximate ratio of activity with lecithin, glucose 6-phosphate, and sodium β-glycerophosphate is 11 : 78.5 : 100. The energies of activation for the hydrolysis of the three substrates do not differ appreciably and the average energy of activation is 14,500 calories per gram-mole. The similarity of the energies of activation together with results from inhibition studies indicate that in all probability the same enzyme is responsible for the release of inorganic phosphorus from each of the three substrates.

scholarly journals Organic pyrophosphates as substrates for human alkaline phosphatases

1967 ◽  
Vol 105 (3) ◽  
pp. 1307-1312 ◽  
Author(s):  
R. Helen Eaton ◽  
D W Moss
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Inorganic Phosphate ◽  
Starch Gel Electrophoresis ◽  
Alkaline Phosphatases ◽  
Starch Gel ◽  
Pyrophosphatase Activity ◽  
Small Intestinal ◽  
Hydrolysis Of ◽  
Single Enzyme

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg2+ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.

scholarly journals Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines

1983 ◽  
Vol 211 (3) ◽  
pp. 553-558 ◽  
Author(s):  
C M Behrens ◽  
C A Enns ◽  
H H Sussman
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Line ◽  
Tumour Cell ◽  
Tumour Cell Line ◽  
Subunit Structure ◽  
Human Tumour ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Inhibition Studies

The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line.

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