Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer

Abstract

The functional unit molecular size of the intestinal brush border membrane-bound Na+/glucose cotransporter was determined by radiation inactivation. Purified brush border membrane vesicles preserved in cryoprotectant buffer were irradiated (-135 degrees C) with high-energy electrons from a 13-MeV (1 eV = 1.602 x 10(-19) J) linear accelerator at doses from 0 to 70 Mrad (1 rad = 0.01 Gy). After each dose, the cotransporter was investigated with respect to (i) Na(+)-dependent transport activity and (ii) immunologic blot analysis with antibodies against the cloned rabbit intestinal cotransporter. Increasing radiation decreased the maximal Na(+)-dependent cotransporter activity Jmax without affecting apparent Km. The size of the transporting functional unit was 290 +/- 5 kDa. Immunologic blot analysis of brush border membranes gave a single band of Mr 70,000, which decreased in intensity with increased radiation dose and gave a target size of 66 +/- 11 kDa. We conclude that activity of the intestinal Na+/glucose cotransporter in situ in the brush border membrane requires the simultaneous presence of four intact, independent, identical subunits arranged as a homotetramer.