AFFINITY IDENTIFICATION OF ORGANIC ANION TRANSPORTERS IN BRUSH-BORDERMEMBRANE-VESICLES FROM RAT-KIDNEY

The inhibitory properties of bromoacetyl-p-aminohippuric acid as the a ffinity probe of the organic anion transport system were studied. Brom oacetylated p-aminohippurate was shown to be able to inhibit irreversi bly the p-aminohippurate (PAH) uptake in brush-border membrane vesicle s. The inhibition depends on both the time of treatment and the affini ty probe concentration. The treatment of brush-border membrane with 1 mM bromoacetyl-p-aminohippurate for 1.5 h results in 100% irreversible inhibition of PAH transport but no changes were observed in the activ ity of alkaline phosphatase, gamma-glutamyltranspeptidase or maltase. The affinity labelling of the organic anion transporters was performed with bromoacetyl-p-amino[H-3]hippuric acid. It was shown, by means of SDS-polyacrylalnide gel electrophoresis, that the probe bound covalen tly to the brush-border membrane proteins with molecular masses of 28 kDa, 63 kDa, 98 kDa, and > 150 kDa. The data obtained with SITS and pr obenecide as the organic anion transport inhibitors indicate that brus h-border membrane proteins of 28 kDa, 63 kDa, 98 kDa may correspond to the organic anion transport system.