Bound and purified alkaline phosphatase from rat duodenal and jejunal brush-border membranes: Kinetics and magnesium stimulation

Abstract

Brush-border membranes from rat duodenal and jejunal mucosa were prepared by differential Ca²⁺-precipitation. Kinetical properties and Mg²⁺-stimulation of alkaline phosphatase were studied for the enzyme either bound to these membranes or purified from these membranes by liquid chromatography.

With p-nitrophenylphosphate as substrate, the alkaline phosphatase apparent K_m was lower in jejunum (90 μM) as compared with duodenum (160 μM), and lower for the purified enzyme (jejunum : 55 μM; duodenum : 97 μM) as compared to the bound one. In the presence of 5 mM MgCl₂, the substrate affinity was in all cases decreased.

For the bound enzyme V_max was 10 times greater in duodenum compared to jejunum. 5 mM MgCl₂ tripled the V_max of the duodenal bound enzyme and increased it by 50% for the jejunal one, but a sevenfold increase was recorded for the purified enzyme at both levels of intestine.

The apparent affinity for Mg²⁺ was similar for the bound and the free enzyme, for duodenum and for jejunum (Mg_0.5 : ± 40 μM).