Comparison of the effects of certain thiol reagents on alanine transport in plasma membrane vesicles from rat liver and their use in identifying the alanine carrier

Hayes, M.; McGivan, J D

doi:10.1042/bj2140489

Cited by 17 publications

(12 citation statements)

References 31 publications

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“…Hepatoma cell lines show system A transporters with altered sensitivity to sulfhydryl-modifying reagents [14,29], thus, we wondered whether the activity that develops during liver cell proliferation after partial hepatectomy could have any similarity with the one present in hepatoma cells. Sensitivity of system A to agents like NEM results in irreversible inactivation, which can be protected by the presence of system A substrate [14,29,30], thus supporting the idea that -SH groups are essential for substrate recognition and translocation. Altered sensitivity to NEM was consistent with carriers structurally different in hepatoma cells.…”

Section: Nem (Mm1mentioning

confidence: 68%

Up‐regulation of system A activity in the regenerating rat liver

et al. 1993

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System A activity for neutral amino acid transport, measured as the MeAIB‐sensitive Na+‐dependent l‐alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na+‐dependent transporters, like system ASC (MeAIB‐insensitive Na+‐dependent l‐alanine transport) and the nucleoside carrier show similar inductions. Up‐regulation of system A is not explained by changes in the dissipation rate of the Na+ transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N‐ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up‐regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na+ transmembrane gradient.

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Section: Nem (Mm1mentioning

confidence: 68%

Up‐regulation of system A activity in the regenerating rat liver

et al. 1993

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“…Inhibition of system A and ASC transport activities by NEM It has previously been reported that NEM inhibits alanine uptake by plasma-membrane vesicles from rat liver (Sips & Van Dam, 1981;Hayes & McGivan, 1983). It has also been reported that the thiol-modifying reagent p-chloromercuribenzenesulphonate has a direct effect on a-aminoisobutyric acid (AIB) transport in several hepatoma cell lines (Dudeck et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

“…In the presence of high concentrations of substrates (10 mM-L-alanine+ 100 mM-NaCI), the inhibition of Na+-dependent alanine uptake induced by 10 min of exposure to 30 mM-IA was substantially slowed down (Table 5). Under these conditions, the presence of substrates during the exposure of membranes to IA partially protected system A and ASC transport activities ( previous observations regarding the protective effect of L-alanine on NEM-dependent blocking of alanine transport in rat liver plasma membranes (Hayes & McGivan, 1983). In addition, a direct effect of p-chloromercuribenzene sulphonate, another sulphydryl-modifying reagent, has been reported on AIB transport (A + ASC) in membrane vesicles from H4 hepatoma cells (Dudeck et al, 1987).…”

Section: Na+-dependentmentioning

confidence: 99%

“…There is evidence that the system A carrier is a glycoprotein that contains an N-linked oligosaccharide moiety (Barber et al, 1983;Kilberg et al, 1985) and shows affinity to concanavalin A (Quesada & McGivan, 1988). Furthermore, a 20 kDa protein has been tentatively identified as an essential component of alanine carriers (A or ASC) in plasma membrane from rat liver (Hayes & McGivan, 1983).…”

Section: Introductionmentioning

confidence: 99%

“…System A and ASC carriers in hepatocytes or plasmamembranes vesicles derived from rat liver are sensitive to thiolgroup-modifying reagents (Kilberg et al, 1980;Sips & Van Dam, 1981;Hayes & McGivan, 1983;Chiles et al, 1988). In the present study, in order to gain further structural information on systems A and ASC, we have investigated the sensitivity of both carrier systems in rat liver plasma-membrane vesicles to chemical modification induced by the thiol-group-modifying reagents Nethylmaleimide (NEM) and iodoacetamide (IA).…”

Section: Introductionmentioning

confidence: 99%

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Sensitivity of system A and ASC transport activities to thiol-group-modifying reagents in rat liver plasma-membrane vesicles. Evidence for a direct binding of N-ethylmaleimide and iodoacetamide on A and ASC carriers

Pola

Bertran

Roca

et al. 1990

Biochemical Journal

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1. In the present study we have examined the sensitivity of A and ASC amino-acid-carrier activities in rat liver plasmamembrane vesicles to the thiol-group modifying reagents N-ethylmaleimide (NEM) and iodoacetamide (IA). To this end, the different Na+-dependent entities involved in alanine transport were assessed. 2. NEM inactivated Na+-dependent alanine transport as a result of the inhibition of both system A and ASC transport activities. The functional sensitivity of system A to NEM was greater than that of system ASC. 3. The presence of L-alanine (10 mM) during the exposure of vesicles to NEM afforded partial protection to system A, but not to the ASC, carrier. This effect was specific, since the presence of L-phenylalanine (10 mM) did not cause any protection. 4. Na+ did not protect A or ASC carriers against NEM inactivation; however, the presence of Na+ (100 mM-NaCl) and L-alanine (10 mM) during the exposure of the vesicles to NEM protected against inactivation of system A and ASC transport activities. The extent of protection was greater in the case of the system ASC transport activity than in the case of the A carrier. 5. IA also diminished Na+-dependent alanine transport by inhibition of A and ASC transport activities. Sodium and L-alanine afforded protection to both A and ASC transport activities from the inhibitory action of IA. The extent of protection induced by substrates was similar for both carriers. 6. It is concluded that there is one, or several, free thiol groups in A and ASC carriers, the integrity of which is essential for transport activity. Sensitivity to thiol-group-specific reagents and the pattern of protection with substrates against inactivation is different in A and ASC carriers. That suggests the existence of topological dissimilarities regarding the thiol-group containing site(s) in A and ASC amino acid carriers.

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System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents

Chiles

Kilberg

1986

Journal Cellular Physiology

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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.

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Comparison of the effects of certain thiol reagents on alanine transport in plasma membrane vesicles from rat liver and their use in identifying the alanine carrier

Cited by 17 publications

References 31 publications

Up‐regulation of system A activity in the regenerating rat liver

Up‐regulation of system A activity in the regenerating rat liver

Sensitivity of system A and ASC transport activities to thiol-group-modifying reagents in rat liver plasma-membrane vesicles. Evidence for a direct binding of N-ethylmaleimide and iodoacetamide on A and ASC carriers

System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents

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