Evidence for inherent differences in the system A carrier from normal and transformed liver tissue. Differential inactivation and substrate protection in membrane vesicles and reconstituted proteoliposomes.
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.
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D026905:Amino Acid Transport Systems; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D009646:Norleucine / Q000494:pharmacology; D051381:Rats
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D008081:Liposomes; D002731:4-Chloromercuribenzenesulfonate; D009646:Norleucine; D005033:Ethylmaleimide