Effect of magnesium on ATP labelling by kidney brush border membrane

1. Brush border membranes purified from rat kidney cortex were incubated in the presence of ATP and analysed by SDS polyacrylamide gel electrophoresis. 2. Quantitative analysis of phosphorylation was performed with a calibration curve obtained by autoradiography. 3. The presence of magnesium was required for the phosphorylation of membrane proteins. 4. EDTA completely inhibited the labelling of all bands, except for the alkaline phosphatase band. 5. In contrast, alkaline phosphatase was inhibited by 52, 65 and 85% in the presence of 1 mM bromotetramisole, 10 mM NaF and 10 mM Na arsenate respectively. 6. However these inhibitors had only minor effects on the labelling of other proteins. 7. High concentrations of magnesium caused a pronounced inhibition on the labelling of the alkaline phosphatase band but had no effect on the phosphorylation of other proteins.