Enzymatic and transport characteristics of isolated snake renal brush-border membranes

Abstract

Brush-border membranes (BBM) of proximal tubules were isolated from the kidney of the garter snake (Thamnophis sirtalis) by a procedure involving hypotonic lysis, Ca precipitation, and differential centrifugation. The isolated membranes were enriched 15-fold in brush-border enzyme activities (alkaline phosphatase, gamma-glutamyl transpeptidase) compared with whole kidney homogenates and were substantially free of other contaminating membranes. The yield of the BBM preparation was 40%. The BBM vesicular transport of several organic solutes was characterized by a rapid filtration technique at 25 degrees C. D-glucose, p-aminohippurate (PAH), and urate entered the same osmotically active space (2-3 microliter/mg protein) and binding was minimal (less than 20% for PAH). An uptake overshoot for 3-O-methyl-D-glucose (20 microM) by reptilian BBM was observed only in the presence of an inwardly directed NaCl gradient and was abolished by 0.1 mM phlorizin. Reptilian BBM exhibited Na-gradient-stimulated uptake of PAH (90 microM) with an overshoot that was inhibited by other organic acids and by 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid (SITS). In contrast, urate uptake (30 microM) appeared to be Na independent and not appreciably affected by other organic anions or SITS. The presence of specific transport systems for organic solutes in the isolated membrane preparation distinctly characterizes the BBM of reptilian kidney.