Intestinal brush border membrane Na+/glucose cotransporter functions in situ as a homotetramer.
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.
Stevens
B R
BR
Department of Physiology, College of Medicine, University of Florida, Gainesville 32610-0274.
Fernandez
A
A
Hirayama
B
B
Wright
E M
EM
Kempner
E S
ES
D000818:Animals; D015153:Blotting, Western; D004307:Dose-Response Relationship, Radiation; D007421:Intestine, Small / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D008961:Models, Structural; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D011817:Rabbits
D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins