Differential Inactivation of System A in Normal and Transformed Liver Cells

Abstract

System A-mediated neutral amino acid transport into normal rat hepatocytes and several hepatoma cell lines was tested for inactivation by sulfhydryl-preferring protein-modifying reagents such as N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). System A activity in hepatocytes and four different hepatoma cell lines was equally sensitive to inhibition by the organic mercurial PCMBS. Inactivation by NEM was substantially different, normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids with a high affinity for System A protected the transporter from inactivation in a stereospecific and systemspecific manner, whereas non-substrates afforded little or no protection. The substrate-dependent protection from PCMBS inactivation was observed in hepatoma cells, but not in normal rat hepatocytes. The differential sensitivity to NEM was also observed when plasma membrane vesicles were prepared from hepatocytes and hepatoma cells and then treated with PCMBS or NEM.