The transport of
amino acids by normal rat hepatocytes and several
hepatoma cell lines has been examined for inactivation by various
protein-modifying
reagents, including the sulfhydryl-preferring
reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (
PCMBS). Uptake of
2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial
PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the
hepatoma cells varied in their responsiveness from moderate to no inhibition.
PCMBS inactivated greater than 85% of the System A activity in rat H4
hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by
PCMBS was rapidly reversed by treatment of the cells with
dithiothreitol.
Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection.
Amino acid-dependent protection was stereospecific and system-specific. L-
norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from
PCMBS-dependent inactivation (half-maximal protection occurred at an
amino acid concentration of 0.6 +/- 0.1 mM).
N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat
hepatoma cells with this
reagent resulted in greater than 95% inhibition.