Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes

• Published in: The Journal of biological chemistry Vol. 265; no. 23; pp. 13914 - 13917
• Main Authors: TARNUZZER, R. W, CAMPA, M. J, QIAN, N.-X, ENGLESBERG, E, KILBERG, M. S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.08.1990
• Subjects: Aminoisobutyric Acids - metabolism; Animals; Biological and medical sciences; Carrier Proteins - genetics; Cell Line; Female; Fundamental and applied biological sciences. Psychology; Gene expression; Humans; Kinetics; Liver - metabolism; Microinjections; Molecular and cellular biology; Molecular genetics; Oocytes - metabolism; Placenta - metabolism; Pregnancy; Rats; RNA, Messenger - administration & dosage; RNA, Messenger - genetics; RNA, Messenger - isolation & purification; system A; Xenopus laevis; Heterologous system; Xenopus laevis; Amphibia; Salientia; Gene expression; Biological activity; Vertebrata; Mammalia; Microinjection; Messenger RNA; Aminoacid; Oocyte; Carrier protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)77435-8

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone. The degree of expression of the mammalian System A activity in Xenopus oocytes corresponded to the known transport rates in the tissue from which the mRNA was prepared. For example, hepatic mRNA from glucagon-treated rats produced greater System A activity than mRNA from control animals, and the mRNA from the CHO transport mutant cell line alar4-H3.9, which overproduces System A, resulted in higher transport rates than mRNA from the parental cell line (CHO-K1). Fractionation of total mRNA poly(A+) by nondenaturing agarose gel electrophoresis revealed transport activity associated with a 2.0-2.5-kilobase mRNA fraction common to each of the three tissues tested.