Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants.
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.
D000409:Alanine / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004351:Drug Resistance; D005784:Gene Amplification; D005796:Genes; D007700:Kinetics; D009154:Mutation; D012964:Sodium / Q000494:pharmacology
D000596:Amino Acids; D012964:Sodium; D000409:Alanine