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The action of cyanate on human and pig kidney alkaline phosphatases.

ABSTRACT: 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 V(max.) and increased K(m).

SUBMITTER: Carey MJ

PROVIDER: S-EPMC1187603 | biostudies-other | 1969 Mar

REPOSITORIES: biostudies-other

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Human kidney and urinary alkaline phosphatases.

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The reversible inactivation of pig kidney alkaline phosphatase at low pH.

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Alkaline phosphatase from pig kidney. Method of purification and molecular properties.

Project description:Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH(4))(2)SO(4) precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000-156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25 degrees C of 2600s(-1) per tetramer. Its concentration in kidney was estimated to be 8.5-8.8mg/kg.

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Evidence for the importance of arginine residues in pig kidney alkaline phosphatase.

Project description:The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

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Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid.

Project description:Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg(2+) and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of K(m) and V(max.) were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.

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