Amino Acid Transport in Membrane Vesicles from CHO-K1 and Alanine-resistant Transport Mutants

Overview

Journal Biochemistry

Specialty Biochemistry

Date 1987 May 5

PMID 3607029

Citations 5

Authors

J Moffett

M Jones

E ENGLESBERG

Affiliations

Soon will be listed here.

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.

Citing Articles

Enhancement in amount of P1 (hsp60) in mutants of Chinese hamster ovary (CHO-K1) cells exhibiting increases in the A system of amino acid transport.

Jones M, Gupta R, ENGLESBERG E Proc Natl Acad Sci U S A. 1994; 91(3):858-62.

PMID: 7905632 PMC: 521411. DOI: 10.1073/pnas.91.3.858.

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.

Moffett J, Perier F, Jones M, ENGLESBERG E Proc Natl Acad Sci U S A. 1987; 84(22):8040-3.

PMID: 3317400 PMC: 299472. DOI: 10.1073/pnas.84.22.8040.

Neutral amino acid transport systems in animal cells: potential targets of oncogene action and regulators of cellular growth.

Saier Jr M, Daniels G, Boerner P, Lin J J Membr Biol. 1988; 104(1):1-20.

PMID: 3054116 DOI: 10.1007/BF01871898.

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.

Qian N, Jones M, McDonough A, ENGLESBERG E Proc Natl Acad Sci U S A. 1989; 86(20):7984-8.

PMID: 2554299 PMC: 298197. DOI: 10.1073/pnas.86.20.7984.

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.

Qian N, Pastor-Anglada M, ENGLESBERG E Proc Natl Acad Sci U S A. 1991; 88(8):3416-20.

PMID: 1849656 PMC: 51458. DOI: 10.1073/pnas.88.8.3416.