BOUND AND PURIFIED ALKALINE-PHOSPHATASE FROM RAT DUODENAL AND JEJUNAL BRUSH-BORDER MEMBRANES - KINETICS AND MAGNESIUM STIMULATION

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-mu-M) as compared with duodenum (160-mu-M), and lower for the purified enzyme (jejunum : 55-mu-M; duodenum : 97-mu-M) 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 seven-fold 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 (Mg0.5 : +/- 40-mu-M).