Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes

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Abstract
Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na+-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na+-dependent α-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained ~ 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 μM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 μM p-chloromercuribenzenesulfonic acid inhibited Na+-dependent α-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+. Transport activity was also inhibited by treatment of solubilized membranes with dithiothreitol, suggesting that the amino acid carrier contains an essential disulfide bridge. p-Chloromercuribenzenesulfonic acid inhibited transport activity could be partly recovered by 2-mercaptoethanol or 2,3-dimercapto-1-propanol, with a maximum recovery of 65% of the original activity at 20 mM of the dithiol.Key words: reconstitution, Na+-dependent amino acid transport, p-chloromercuribenzenesulfonic acid inhibition, substrate protection.