木瓜蛋白酶对38,000道尔顿片段的切割抑制了4,4 ' -二异硫氰基二苯乙烯- 2,2 ' -二磺酸盐与人带3中60,000道尔顿片段上的赖氨酸-539的结合

Takeo Yamaguchi, Hideaki Kojima, Shiori Kawaguchi, M. Shimada, Haruka Aso
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引用次数: 1

摘要

Human band 3是一个98 kda的跨膜蛋白(TM),由14个TM片段组成。木瓜蛋白酶将能带3切割成38和60 kda的片段。在强力条件下,38-kDa片段中门域TM 7和核心域TM 8之间的环区被切割产生7-和31-kDa片段。含有Lys-539的TM -5片段在38-kDa片段切割后的构象变化尚不清楚。4,4 '-二异硫氰基二苯乙烯- 2,2 '-二磺酸盐(DIDS)与Lys-539结合可抑制压力诱导的红细胞溶血。由于抑制了DIDS与Lys-539的结合,因此在38-kDa片段的切割中没有观察到DIDS的这种作用。利用标记为Lys-539的DIDS荧光检测了3波段的构象变化。荧光光谱表明,DIDS的分子运动在38-kDa片段的消化过程中受到更大的限制。有趣的是,DIDS荧光猝灭表明,38-kDa片段被DIDS消化后,Hg2+更难被DIDS获取。综上所述,我们认为以Lys-539的隔离和受限运动为特征的tm5片段的构象变化是由tm7和tm8之间的环区断裂引起的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Papain cleavage of the 38,000-dalton fragment inhibits the binding of 4, 4′-diisothiocyanostilbene-2, 2′-disulfonate to lys-539 on the 60,000-dalton fragment in human band 3
Human band 3 is a 98-kDa transmembrane (TM) protein comprising 14 TM segments. Papain cleavages band 3 into 38- and 60-kDa fragments. Under vigorous conditions, the cleavage of the loop region between the TM 7 of gate domain and the TM 8 of core domain in the 38-kDa fragment produces 7- and 31-kDa fragments. Conformational changes of the TM 5 segment containing Lys-539 by cleavage of the 38-kDa fragment remain unclear. Pressure-induced haemolysis of erythrocytes was suppressed by binding of 4, 4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) to Lys-539. Such effect of DIDS was not observed upon cleavage of the 38-kDa fragment, because of inhibition of DIDS binding to Lys-539. Using fluorescence of DIDS labelled to Lys-539, conformational changes of band 3 were examined. Fluorescence spectra demonstrated that the molecular motion of DIDS is more restricted upon digestion of the 38-kDa fragment. Interestingly, the quenching of DIDS fluorescence showed that Hg2+ is less accessible to DIDS upon digestion of the 38-kDa fragment. Taken together, we propose that the conformational changes of the TM 5 segment characterized by the sequestration and restricted motion of Lys-539 are induced by the cleavage of the loop region between the TM 7 and the TM 8.
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