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The pyrophosphatase activity of pig kidney alkaline phosphatase and its inhibition by magnesium ions and excess of pyrophosphate.

ABSTRACT: 1. Pig kidney enzyme resembles other non-specific alkaline phosphatases in its ability to hydrolyse inorganic pyrophosphate (PP(i)). 2. Studies of enzyme velocity as a function of PP(i) concentration show that Michaelis-Menten kinetics are obeyed when a constant PP(i)/Mg(2+) concentration ratio is maintained, but velocity-substrate concentration curves are sigmoid when the concentration of PP(i) is increased but that of Mg(2+) is kept constant. The enzyme is inhibited when the total PP(i) concentration is greater than the total concentration of Mg(2+). Pyrophosphatase activity is activated by Mg(2+), but if the concentration of the metal ion is increased to a value in excess of the total PP(i) concentration Mg(2+) is then strongly inhibitory. 4. It appears that the enzyme is most active towards the complex ion MgPP(i) (2-). The enzyme probably hydrolyses PP(i) (4-) also, but this is a poorer substrate and its competition with MgPP(i) (2-) leads to inhibition. At high Mg(2+) concentrations Mg(2)PP(i) is formed. This complex appears to be a potent inhibitor. 5. Sigmoid plots of v against s and of v against i result from interactions occurring between Mg(2+) and PP(i) (4-) leading to MgPP(i) (2-) and Mg(2)PP(i), and are not indicative of allosteric behaviour.

SUBMITTER: Butterworth PJ

PROVIDER: S-EPMC1187438 | biostudies-other | 1968 Dec

REPOSITORIES: biostudies-other

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Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions.

Project description:The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg(2+) ions was investigated with the aid of kinetic measurements. Mg(2+) ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg(2+) ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg(2+) must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg(2+) ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg(2+), it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

| S-EPMC1168072 | biostudies-other

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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.

| S-EPMC1168074 | biostudies-other

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