The use of HgCl2 to evaluate the cosubstrate: Amino acid transport stoichiometry in ehrlich ascites tumor cells

✍ Scribed by W. David Dawson; Susan C. Robinson; Thomas C. Smith

• Publisher: John Wiley and Sons
• Year: 1983
• Tongue: English
• Weight: 688 KB
• Volume: 115
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041150205

✦ Synopsis

Abstract

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na^+^ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na^+^ and α‐aminoisobutyric acid (α‐AIB) cotransport in Ehrlich ascites tumor cells depleted of Na^+^ and ATP by incubation in Na^+^ ‐free HEPES‐buffered medium (pH 7.2) containing 160 mM K^+^ and 2.5 μM valinomycin. Transfer of the cells to a medium with 10 mM ^22^Na^+^, 10 mM ^3^H‐AIB, and 150 mM K^+^ resulted in an enhancement of Na^+^ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, −7.0 ± 0.1 mV (SEM), does not change with the addition of AIB, −7.3 ± 0.6 mV (SEM). HgCl~2~ (10 m̈M) added to the medium inhibited AIB flux and AIB‐stimulated Na^+^ flux by 45–50% but did not change the coupling ratio. HgCl~2~ (10 m̈M) does not inhibit the basal Na^+^ flux nor does it affect cellular Na^+^ or K^+^ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is −22.3 ± 0.8 mV (SEM) and depolarizes to −16.7 ± 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 ± 0.02 (SEM) in the presence of HgCl~2~ (10 m̈M). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of α‐AIB. We suggest that H^+^ provides the alternative cosubstrate in this low Na^+^ environment and that in high Na^+^ medium the Na^+^ :AIB stoichiometry approaches 1:1.