Plasma membrane vesicles isolated from intact rat liver (normal hepatocyte) or cultured rat H4
hepatoma cells retain Na+-dependent uptake of
2-aminoisobutyric acid mediated by System A. The carrier was inactivated in normal liver membrane vesicles by either
N-ethylmaleimide (NEM) or p-chloromercuribenzene sulfonate (
PCMBS). The concentrations required to produce half-maximal inhibition were approximately 370 and 110 microM for NEM and
PCMBS, respectively. In contrast, transport of System A in H4
hepatoma membrane vesicles was sensitive to
PCMBS (K 1/2 = 180 microM), yet totally unaffected by NEM at concentrations up to 5 mM. Substrate-dependent protection from
PCMBS activation was observed for the System A activity in H4
hepatoma membranes, but not in vesicles from normal hepatocytes. Subsequent inactivation of the substrate-protected carrier by sulfhydryl-specific
reagents, added following the removal of the protective
amino acid, suggests that one or more
cysteine residues become less reactive in the presence of System A substrates. Treatment of solubilized
membrane proteins with NEM prior to reconstitution into artificial
proteoliposomes showed that the selective inactivation by NEM of the carrier in normal liver membranes is not dependent on the
lipid environment or on the integrity of the plasma membrane. The results support the hypothesis that there are inherent differences in the System A carriers that are present in normal and transformed liver tissue.