Photoaffinity labeling of organic anion transport system in proximal tubule

Abstract

The present investigation compares brush-border (BBM) and basolateral membrane (BLM) vesicles in terms of purity, function, appearance on sodium dodecyl sulfate (SDS) gels, and labeling pattern by use of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid (NAP-taurine), a photoaffinity analogue of p-aminohippurate (PAH). Both BLM and BBM were vesicular by demonstration of PAH uptake into an osmotically active space and had probenecid-inhibitable uptake of PAH. Time courses for uptake were similar. 250 microM NAP-taurine resulted in a 35% inhibition of PAH uptake in BLM but it did not significantly effect PAH uptake into BBM. The latter was affected by 1 mM NAP-taurine. A comparison of Coomassie blue SDS gels of BLM and BBM showed markedly different staining patterns. A major band at approximately 52,000 daltons was more intensely stained in BLM than BBM. Major bands at approximately 40,000 and approximately 80,000 were stained more heavily in BBM than BLM. A minor protein at 26,000 in the BLM did not appear in BBM. An irreversible inhibition of PAH uptake in BLM was observed after photolysis in the presence of NAP-taurine. This was associated with the labeling of four protein bands on SDS polyacrylamide gels. In contrast no labeling was observed in BBM.