nitrophenols and thiophosphoric-acid

Topics: 5'-Nucleotidase; Esters; Humans; Isomerism; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Conformation; Nitrophenols; Nucleotidases; Nucleotides; Organophosphorus Compounds; Phosphates; Phosphoric Diester Hydrolases

In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors.. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity.. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Topics: Alkaline Phosphatase; Baculoviridae; Clinical Enzyme Tests; Down Syndrome; Enzyme Inhibitors; Enzyme Stability; Female; GPI-Linked Proteins; Humans; Isoenzymes; Kinetics; Neutrophils; Nitrophenols; Organophosphorus Compounds; Phosphates; Placenta; Pregnancy; Protein Denaturation; Tetramisole

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

Topics: Alanine; Alkaline Phosphatase; Arginine; Enzyme Inhibitors; Escherichia coli; Hydrolysis; Kinetics; Linear Energy Transfer; Mutagenesis, Site-Directed; Nitrophenols; Organophosphorus Compounds; Organothiophosphorus Compounds; Phosphates; Substrate Specificity; Thionucleotides

Topics: Aging; Animals; Cholinergic Agents; Guinea Pigs; Insecticides; Liver; Metabolism; Mice; Microsomes; Nitrophenols; Oxidoreductases; Parathion; Phosphates; Rats; Research; Sex; Species Specificity; Toxicology

Topics: Chromatography; Chromatography, Thin Layer; Insecticides; Nitrophenols; Phosphates; Rare Diseases; Research

Topics: Insecticides; Nitrophenols; Phosphates

Topics: Insecticides; Nitrophenols; Occupational Health; Parathion; Phosphates