Cloherty E K, Diamond D L, Heard K S, Carruthers A
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605, USA.
Biochemistry. 1996 Oct 8;35(40):13231-9. doi: 10.1021/bi961208t.
Avian erythrocyte sugar transport is stimulated during anoxia and during exposure to inhibitors of oxidative phosphorylation. This stimulation results from catalytic desuppression of the cell surface glucose transporter GLUT1 [Diamond, D., & Carruthers, A. (1993) J. Biol. Chem. 268, 6437-6444]. The present study was undertaken to investigate the mechanisms of GLUT1 suppression/desuppression. Sugar uniport (sugar uptake or exit in the absence of sugar at the opposite side of the membrane) is absent in normoxic avian erythrocytes, but sugar antiport (sugar uptake coupled to sugar exit) is present. Exposure to cyanide and/or to FCCP (mitochondrial inhibitors) stimulates erythrocyte sugar uniport but not sugar antiport. K(m)(app) for 3-O-methylglucose uniport and antiport are unaffected by metabolic poisoning. Ki(app) for inhibitions of 3-O-methylglucose uniport by cytochalasin B and forskolin (sugar export site ligands) are unaffected by progressive stimulation of sugar uniport. Cyanide and FCCP stimulation of 3-O-methylglucose uniport are associated with increased AMP-activated protein kinase activity. Purified human GLUT1 is not phosphorylated by exposure to cytosol extracted from poisoned avian erythrocytes. FCCP does not stimulate GLUT1-mediated 3-O-methylglucose uptake in K562 cells but does increase K562 AMP-activated protein kinase activity. FCCP stimulation of 3-O-methylglucose uniport in resealed erythrocyte ghosts requires cytosolic ATP and/or glutathione. The nonmetabolizable ATP analog AMP-PNP cannot be substituted for ATP in this action. These results are contrasted with allosteric regulation of human erythrocyte sugar transport and suggest that avian erythrocyte sugar transport suppression results from inhibition of carrier uniport function. Uniport suppression is not mediated by interaction with cytosolic molecular species that bind to the sugar export site. The antiport to uniport switch mechanism requires ATP hydrolysis, is associated with elevated AMP-activated kinase function, and, if triggered by this kinase, is mediated by factors absent in K562 cells and downstream from the kinase.
缺氧期间以及暴露于氧化磷酸化抑制剂时，禽类红细胞的糖转运受到刺激。这种刺激源于细胞表面葡萄糖转运蛋白GLUT1的催化去抑制作用[戴蒙德，D.，& 卡拉瑟斯，A.（1993年）《生物化学杂志》268卷，6437 - 6444页]。本研究旨在探究GLUT1抑制/去抑制的机制。在常氧禽类红细胞中不存在糖单向转运（在膜的另一侧无糖存在时的糖摄取或流出），但存在糖反向转运（糖摄取与糖流出偶联）。暴露于氰化物和/或FCCP（线粒体抑制剂）会刺激红细胞糖单向转运，但不刺激糖反向转运。3 - O - 甲基葡萄糖单向转运和反向转运的表观米氏常数（K(m)(app)）不受代谢中毒影响。细胞松弛素B和福斯可林（糖输出位点配体）对3 - O - 甲基葡萄糖单向转运抑制的表观抑制常数（Ki(app)）不受糖单向转运逐渐刺激的影响。氰化物和FCCP对3 - O - 甲基葡萄糖单向转运的刺激与AMP活化蛋白激酶活性增加有关。纯化的人GLUT1暴露于从中毒禽类红细胞中提取的胞质溶胶时不会被磷酸化。FCCP不会刺激K562细胞中GLUT1介导的3 - O - 甲基葡萄糖摄取，但会增加K562细胞中AMP活化蛋白激酶的活性。FCCP对重封红细胞血影中3 - O - 甲基葡萄糖单向转运的刺激需要胞质ATP和/或谷胱甘肽。在此作用中，不可代谢的ATP类似物AMP - PNP不能替代ATP。这些结果与人类红细胞糖转运的变构调节形成对比，并表明禽类红细胞糖转运抑制是由于载体单向转运功能受到抑制。单向转运抑制不是由与结合到糖输出位点的胞质分子物种相互作用介导的。从反向转运到单向转运的转换机制需要ATP水解，与升高的AMP活化激酶功能相关，并且如果由该激酶触发，则由K562细胞中不存在且在激酶下游的因子介导。
