The involvement of the membrane oxidoreduction system in stimulating amino acid uptake in ehrlich ascites tumor cells

Abstract

The addition of 5 mM ascorbate plus 0.09 mM phenazine methosulfate stimulated 2− to 3−fold the initial rate of 2-aminoisobutyric acid transport into Ehrlich cells. This was observed under the conditions in which glycolysis and mitochondrial electron transport were blocked by iodoacetate and KCN, and the cellular ATP level was maintained below 0.1 mM. Proton conductors, carbonylcyanide m- chlorophenylhydrazone and SF6847 did not affect the stimulation of 2-aminoisobutyric acid uptake caused by ascorbate plus phenazine methosulfate. Ascorbate was replaced by NADH but not by NADPH, and phenazine methosulfate was the only effective acceptor in stimulating 2-aminoisobutyric acid uptake. The stimulating effect of ascorbate plus phenazine methosulfate was due to an increase in the V value for 2-aminoisobutyric acid but not in the K m value. This effect required the presence of an Na + gradient and was accompanied by an increase in 22Na + influx. The molar ratio of 2-aminoisobutyric acid to Na + uptake enhanced by ascorbate plus phenazine methosulfate was calculated to be 1 : 1. Quinacrine, an inhibitor of NADH oxidoreductase in the plasma membrane, inhibited both the enhanced rate of 2-aminoisobutyric acid and Na + transport without affecting the basal transport activity. The stimulatory effect of ascorbate plus phenazine methosulfate was also observed with other amino acids, alanine, glycine, proline and cycloleucine which are known to be transported via an Na + dependent system but not with leucine and threonine. These results suggest that a redox system in the plasma membrane participates in energy coupling for amino acid transport by increasing the rate of cotransport with Na +.