Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.
Cytochalasin B interferes with conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding.
复制标题
细胞松弛素 B 干扰由内部和外部糖结合引起的人红细胞葡萄糖转运蛋白的构象变化。
DOI：
10.1021/bi00113a009
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Carter-Su,C
中科院分区：
文献类型：
--
作者：
King,AP;Tai,PK;Carter-Su,C
Anthony PJ King，Ping-Kaung Ku Tai * 和Christin Carter-Su* 密歇根大学医学院生理学系，安阿伯，密歇根州48109-0622接收于1991年2月6日;修订版Mandarin pt接收于1991年9月13日摘要：为了深入了解易化糖转运的机制和可能改变葡萄糖转运蛋白内在活性的可能机制，我们研究了由内部和外部糖结合以及由转运抑制剂细胞松弛素B诱导的人红细胞葡萄糖转运蛋白的构象变化。嗜热菌蛋白酶抑制红细胞血影中存在的葡萄糖转运蛋白的能力的变化被用来监测葡萄糖转运蛋白的构象变化。蛋白酶消化的程度通过蛋白酶处理后剩余的未消化的葡萄糖转运蛋白的量来确定，如使用葡萄糖转运蛋白特异性单克隆抗体7 F7在Western印迹中评估的。5. D-葡萄糖，转运蛋白的生理底物，增加转运蛋白的敏感性裂解嗜热菌蛋白酶。特异性结合到内部或外部葡萄糖转运蛋白糖结合位点的非转运葡萄糖类似物也改变了转运蛋白对嗜热菌蛋白酶的敏感性。甲基和丙基葡糖苷，优先结合内部糖位点，增加嗜热菌蛋白酶的敏感性的葡萄糖转运蛋白的方式类似的D-葡萄糖。相反，4，6-O亚乙基葡萄糖，它优先结合外部糖位点，保护转运蛋白免受嗜热菌蛋白酶消化。这些结果表明，糖结合到内部和外部的糖网站诱导不同的构象变化和观察到的D-葡萄糖对葡萄糖转运蛋白的嗜热菌蛋白酶的易感性的影响是由于D-葡萄糖在平衡主要形成一个复杂的内部糖网站。由外部糖结合引起的对嗜热菌蛋白酶裂解的保护作用通过加入内部结合糖而减弱。细胞松弛素B在10 μ M的浓度下，最大限度地抑制转运，对转运蛋白水解没有影响，与外部或内部结合糖的影响形成鲜明对比。细胞松弛素B可阻断亚乙基葡萄糖的保护作用以及D-葡萄糖和甲基葡萄糖苷对转运蛋白水解的增强作用。因此，细胞松弛素B不诱导与糖结合到任一位点相同的构象变化。细胞松弛素B不是将葡萄糖转运蛋白锁定为向内或向外的构象，而是通过将转运蛋白锁定为阻止诱导糖诱导构象的构象来抑制转运。这些数据反对一个三元复合物的转运与外部结合的糖和细胞松弛素B。促进扩散的糖跨膜的哺乳动物细胞是由特定的跨膜转运蛋白，葡萄糖转运蛋白介导的。这些蛋白质中的几种现已被克隆（Mueckler et al.，1985; Kayano等人，1988; Birnbaum等人，1989; Fukumoto等人，1989; James等人，1989 a），并显示出高度的残基相似性，特别是在跨膜区。因此，预计它们具有类似的运输机制。鉴于最近的证据表明细胞膜中葡萄糖转运蛋白的内在活性可能受到胰岛素和其他激素如β-肾上腺素能激动剂异丙肾上腺素的调节，理解这种机制是特别重要的（Joost et al.，1986;黑田等人，1987 …
Anthony PJ King, Ping-Kaung Ku Tai,* and Christin Carter-Su* Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622 Received February 6, 1991; Revised Manuscript Received September 13, 1991 abstract: To gain insight into the mechanism of facilitated sugar transport and possible mechanisms by which glucose transporter intrinsic activity might be altered, we have investigated conformational changes of the human erythrocyte glucose transporter induced by internal and external sugar binding and by the transport inhibitor, cytochalasin B. Changes in the ability of thermolysin to digestglucose transporters present in erythrocyte ghosts were used to monitor conformational changes of the glucose transporter. The degree of protease digestion was determined by the amount of undigested glucose transporter remaining after theprotease treatment, as assessed in Western blots using theglucose transporter specific monoclonal antibody 7F7. 5. D-Glucose, the physiological substrate of the transporter, increased the transporter’s susceptibility tocleavage by thermolysin. Nontransportable glucose analogues which bind specifically to either an internal or external glucose transporter sugar binding site also altered susceptibility of the transporter to thermolysin. Both methyl and propyl glucoside, which preferentially bind the internal sugarsite, increased thermolysin susceptibility of the glucose transporter in a manner similar to that of D-glucose. In contrast, 4, 6-Oethylideneglucose, which preferentially binds the external sugar site, protected the transporter from thermolysin digestion. These results suggest that sugar binding to internal and external sugar sites induces distinct conformational changes and that theobserved D-glucose effect on the susceptibility of the glucose transporter to thermolysin is dueto D-glucose at equilibrium predominantly forming a complex with the internal sugar site. The protectionfrom cleavage by thermolysin caused by external sugar binding is attenuated by the addition of an internally binding sugar. Cytochalasin B at 10 juM, a concentration that inhibits transport maximally, had no effect on transporter proteolysis, in marked contrastto the effects of externally or internally binding sugars. However, cytochalasin B blocked both the protective effect of ethylideneglucose and the potentiating effect of D-glucose and methyl glucoside on transporter proteolysis. Thus, cytochalasin B does not induce the same conformational change as sugar binding to either site. Rather than locking the glucose transporter into either the inward-or outward-facing conformation, cytochalasin B appears to inhibit transport by locking the transporter into a conformation that prevents induction of either sugar-induced conformation. These data argue against a ternary complex of the transporter with an externally bound sugar and cytochalasin B.The facilitated diffusion of sugars across the membranes of mammalian cells is mediated by specific transmembrane transport proteins, the glucose transporters. Several of these proteins have now been cloned (Mueckler et al., 1985; Kayano et al., 1988; Birnbaum et al., 1989; Fukumoto et al., 1989; James et al., 1989a) and show a high degree of residue sim-ilarity, particularly in the transmembrane regions. Therefore, they are expected to have a similar mechanism of transport. Understanding this mechanism is of particularimportance in light of recent evidence which suggests that the intrinsic ac-tivity of glucose transporters in the cell membrane may be modulated by insulin and other hormones, such as the 0-adrenergic agonist isoproterenol (Joost et al., 1986; Kuroda et al., 1987 …
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DOI：
10.1016/s0021-9258(19)37577-5
发表时间：
1988-10
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
T. Kayano;H. Fukumoto;R. Eddy;Y. Fan;M. Byers;T. Shows;G. Bell
通讯作者：
T. Kayano;H. Fukumoto;R. Eddy;Y. Fan;M. Byers;T. Shows;G. Bell
DOI：
10.1016/0005-2736(71)90045-9
发表时间：
1971-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
GECK, P
通讯作者：
GECK, P
扩散无法解释绵羊胎盘葡萄糖转移并考虑可能的载体转移动力学
DOI：
10.1113/jphysiol.1952.sp004770
发表时间：
1952
期刊：
The Journal of Physiology
影响因子：
--
作者：
W. F. Widdas
通讯作者：
W. F. Widdas
氟二硝基苯使葡萄糖穿过人红细胞膜的转运失活。
DOI：
10.1016/0005-2736(73)90107-7
发表时间：
1973
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
P. Edwards
通讯作者：
P. Edwards
异常运输动力学和葡萄糖载体假说。
DOI：
10.1016/0005-2736(74)90320-4
发表时间：
1974
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
D. Regen;H. L. Tarpley
通讯作者：
H. L. Tarpley