Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants

Abstract

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.

Control of A-system amino acid transport by a second regulatory gene R2 in Chinese hamster ovary cells CHO-K1 and the possible connection of this gene with insulin activity.

“…By stepwise selection with increasing concentrations of alanine, mutants have been isolated from a constitutive mutant, Alar4, that have further increases in A-system transport that may result from gene amplification (J.M., M.J., E. Mendiaz, and E.E., unpublished data). Biologically active membrane vesicles prepared from one of these mutants and Alar4 have greater A-system activity than those prepared from the parent cell line (11). This, together with the identification of two membrane proteins present in greater abundance in the plasma membrane of an "amplified" mutant (J.M., M.J., E. Mendiaz, and E.E., unpublished data), further supports the proposed model of gene regulation.…”

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

“…It is Na' dependent and transports one Na' into the cells with each amino acid (3). This system is inducible by hormones (4)(5)(6), repressible by amino acids, which it generally transports (7)(8)(9)(10)(11), and present with increased activity in many transformed and malignant cells (6).…”

alar4, a constitutive mutant of the A system for amino acid transport, has increased abundance of the Na+,K+-ATPase and mRNA for alpha 1 subunit of this enzyme.

“…We show that alar4, which we have previously shown to have an increase in abundance of the A system (1,3), has an increase in ouabain sensitivity, as measured by efficiency of plating, increase in activity and abundance of the WT Na+,K+-ATPase, over that present in the WT, and an increase in abundance of the mRNA for the al subunit, whereas the Na+ concentration does not differ significantly from that of the WT. As an alternative to Na+ having an effect on the abundance of the Na' pump, our evidence suggests that regulatory gene RI, previously shown to regulate the expression of the gene for the A system transporter (1-6), and perhaps other similar transacting regulatory genes regulate the Na+,K+-ATPase.…”

“…CHO-K1 (WT), alar4, medium employed in this study, MEMCHO-4, and cultural conditions have been described (1,3,7 [20][21][22][23][24][25][26][27][28][29][30] sec. The dishes were allowed to dry, 0.6 ml of 0.1 M NaOH was added, and after 1 hr a sample of 0.4 ml was removed for the determination of radioactivity.…”

“…Ouabain-inhibitable ATPase was determined by measuring the release of inorganic phosphate from ATP (23). Na+,K+-ATPase activity was normalized to the activity of 5'-nucleotidase, a membrane-bound enzyme as described (3).…”