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System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents
Author(s) -
Chiles Thomas C.,
Kilberg Michael S.
Publication year - 1986
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041290309
Subject(s) - dithiothreitol , amino acid , reagent , biochemistry , chemistry , cell culture , norleucine , cell , biology , enzyme , leucine , genetics
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 (t 1/2 = 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 (K i = 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.