Uchida S, Kwon H M, Preston A S, Handler J S
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1991 May 25;266(15):9605-9.
Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.
在注射了从Madin-Darby犬肾（MDCK）细胞中提取的聚腺苷酸（poly(A)+）RNA的非洲爪蟾卵母细胞中，检测了MDCK细胞牛磺酸转运体的表达。与注射水的卵母细胞相比，注射总poly(A)+ RNA导致钠依赖性牛磺酸摄取增加，这与注射的RNA量直接相关。注射poly(A)+ RNA的卵母细胞中的表达量比注射水的卵母细胞高5至10倍。由于在高渗培养基中培养会增加MDCK细胞中牛磺酸摄取的最大速度（Vmax），我们比较了注射来自在高渗培养基中培养的MDCK细胞的poly(A)+ RNA后的卵母细胞牛磺酸摄取与注射来自等渗细胞的poly(A)+ RNA后的卵母细胞牛磺酸摄取，发现高渗细胞的poly(A)+ RNA引起的牛磺酸摄取是等渗细胞的poly(A)+ RNA引起的两倍。在卵母细胞中表达的转运体与MDCK细胞中的类似：它完全依赖于细胞外钠，并且也依赖于阴离子（Cl-≥Br-＞SCN-＞葡萄糖酸盐-）。其他β-氨基酸，β-丙氨酸和亚牛磺酸，抑制牛磺酸摄取，但L-丙氨酸和2-（甲氨基）异丁酸则不抑制。该转运体的表观米氏常数（Km）为7.0微摩尔。在蔗糖密度梯度上进行大小分级分离后，发现编码MDCK牛磺酸转运体的poly(A)+ RNA存在于平均大小为4.4千碱基的级分中。
