Characterization of phosphate binding by alkaline phosphatase in rat kidney brush border membrane.

Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by gamma-32P ATP at 0 degree C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1 +/- 5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein. Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparent Km: one at 40 microM for low and one at 390 microM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.