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
alpha-aminoisobutyric acid transport, while
N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the
N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM
N-ethylmaleimide prior to p-chloromercuribenzenesulfonic
acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic
acid inhibited Na(+)-dependent
alpha-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+.(ABSTRACT TRUNCATED AT 250 WORDS)