Effects of thiol-reactive agents on amino acid transport by sheep erythrocytes.

(1) Sheep erythrocytes possess a facilitated diffusion transport system selective for small neutral amino acids of intermediate size (C-system). The effects of seven thiol-reactive agents on this system were investigated. (2) L-Alanine influx by this route was inhibited by HgCl2, p-chloromercuriphenylsulphonate (PCMBS), azodiacarboxylic acid bisdimethylamide (diamide), N-ethylmaleimide and t-butylhydroperoxide. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoate) had no effect. (3) Detailed analysis of these inhibitor effects suggested the presence of three distinct classes of cellular thiol groups essential for normal transport function. (4) Class 1 thiols react with PCMBS and are located on the outer surface of the cell membrane in the region of the transport site. Class 2 thiols react with N-ethylmaleimide and diamide but are not affected by t-butylhydroperoxide. Class 3 thiols are oxidized during t-butylhydroperoxide treatment and are presumably also attacked by diamide and N-ethylmaleimide. (5) Class 3 thiols are either intracellular GSH or reactive thiols which readily form mixed disulphides with GSSG. Any direct involvement of GSH in amino acid transport is not mediated by the gamma-glutamyl cycle.