Structural state of the Na + / d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na +-dependent phlorizin binding and Na +-dependent d-glucose transport

Abstract

Target sizes of the renal sodium- d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at −50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na +-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na +-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium- d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.