Association of hepatic system A amino acid transporter with the membrane-cytoskeletal proteins ankyrin and fodrin.

System A activity is a highly regulated mechanism for the active transport of zwitterionic amino acids into mammalian cells. Monoclonal antibodies generated against a previously unidentified rat liver plasma membrane-associated protein were shown to immunoprecipitate solubilized System A transport activity. The immunoreactive protein was later determined by immunoblotting and peptide microsequencing to be rat liver alpha-fodrin (non-erythroid spectrin). Antibody against ankyrin, a protein that often serves as a bridge between integral membrane proteins and fodrin, also immunoprecipitated System A transport activity. Fractionation of solubilized plasma membrane proteins on sucrose gradients revealed that the System A transporter co-migrated as a complex with fodrin and ankyrin, even in the presence of detergent and urea. In contrast, the System N amino acid transporter does not co-migrate with ankyrin and fodrin, nor does the anti-fodrin antibody immunoprecipitate System N activity. The present data are the first to demonstrate an association between an organic solute transporter and the membranocytoskeletal proteins ankyrin and fodrin.

Solubilization and reconstitution characteristics of hepatic system A-mediated amino acid transport.

In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 degrees C or for 2 days at 4 degrees C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteoliposomes at specific activities greater than those in the native vesicles.

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.