Gould G W, Lienhard G E
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Biochemistry. 1989 Nov 28;28(24):9447-52. doi: 10.1021/bi00450a030.
A cDNA encoding the rat brain glucose transporter was inserted between the 5' and 3' untranslated regions from the Xenopus globin gene and downstream of an SP6 RNA polymerase start site. RNA synthesized from this vector was microinjected into oocytes from Xenopus laevis; this resulted in expression of the glucose transporter, as determined by both immunoblotting and the appearance of transport activity. The properties of the transporter were those expected from previous studies: it was glycosylated, and its activity, measured by 3-O-methylglucose transport, was inhibited by D-glucose and cytochalasin B, but not by L-glucose. The low level of endogenous glucose transport activity found in water-injected oocytes makes this a useful system in which to determine the kinetic parameters of transport. The Km for 3-O-methylglucose was found to be 20 mM under equilibrium exchange conditions. Despite the fact that oocytes exhibit insulin-dependent responses, insulin did not stimulate 3-O-methylglucose transport by injected oocytes.
将编码大鼠脑葡萄糖转运蛋白的互补DNA（cDNA）插入非洲爪蟾珠蛋白基因的5'和3'非翻译区之间，并置于SP6 RNA聚合酶起始位点的下游。从该载体合成的RNA被显微注射到非洲爪蟾的卵母细胞中；通过免疫印迹和转运活性的出现确定，这导致了葡萄糖转运蛋白的表达。该转运蛋白的特性与先前研究预期的一致：它是糖基化的，通过3-O-甲基葡萄糖转运测量其活性，D-葡萄糖和细胞松弛素B可抑制其活性，但L-葡萄糖无此作用。在注射水的卵母细胞中发现的内源性葡萄糖转运活性水平较低，这使得该系统成为确定转运动力学参数的有用系统。在平衡交换条件下，发现3-O-甲基葡萄糖的米氏常数（Km）为20 mM。尽管卵母细胞表现出胰岛素依赖性反应，但胰岛素并未刺激注射了RNA的卵母细胞对3-O-甲基葡萄糖的转运。
