AMIDINATION OF OUTER AND INNER SURFACES OF HUMAN ERYTHROCYTE-MEMBRANE
AMIDINATION OF OUTER AND INNER SURFACES OF HUMAN ERYTHROCYTE-MEMBRANE
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DOI：
10.1016/0022-2836(74)90103-x
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发表时间：
1974-01-01
影响因子：
5.6
通讯作者：
BERG, HC
中科院分区：
文献类型：
--
作者：
WHITELEY, NM;BERG, HC
我们合成了一种新型亚氨酸酯，即乙酰亚胺羟乙硫酰酯，它无法穿透人红细胞膜。它对氨基具有与乙酰亚胺乙酯相同的特异性，可穿透膜。任一试剂都可以用 3 H或 14 C标记，因此可用于将胺转化为放射性脒。被这两种化合物饱和的红细胞膜的功能几乎正常。因此，如果细胞外表面上的氨基被乙酰亚胺酸乙酯饱和(例如用14 C标记)并且剩余的活性位点被乙酰亚胺乙酯饱和(用3 H标记)，则膜可以被双标记。或者，可以在用[ 14 C]羟乙硫氨酰乙酰亚氨酸酯饱和后分离膜并用[ 3 H]羟乙硫氨酰乙酰亚氨酸酯处理。从此类定量实验中，我们得出结论，膜内表面的蛋白质中的反应性氨基数量是膜外表面的十倍以上。脂质中几乎所有的反应性氨基都在内表面上。单个多肽的定位证实并扩展了先前通过其他技术进行的分配；多达四个主要成分可以跨越膜。蛋白质和脂质在完整细胞中与乙酰亚氨酸乙酯的反应程度与在分离膜中的反应程度相同；这意味着隔离不会导致重大的结构重新安排。
We have synthesized a novel imidoester, isethionyl acetimidate, which is unable to penetrate the membrane of the human erythrocyte. It has the same specificity for amino groups as ethyl acetimidate, which penetrates the membrane. Either reagent can be labeled with3H or14C and, thus, be used to convert amines to radioactive amidines. An erythrocyte membrane saturated with either compound functions nearly normally. Therefore, the membrane can be double labeled if the amino groups on the outer surface of a cell are saturated with isethionyl acetimidate (e.g. labeled with14C) and the remaining active sites are saturated with ethyl acetimidate (labeled with3H). Alternatively, the membrane can be isolated after saturation with [14C]isethionyl acetimidate and treated with [3H]isethionyl acetimidate. From quantitative experiments of this kind we conclude that there are more than ten times as many reactive amino groups in protein on the inner surface than on the outer surface of the membrane. Nearly all of the reactive amino groups in lipid are on the inner surface. The localization of individual polypeptides confirms and extends assignments made previously by other techniques; as many as four major components may span the membrane. The proteins and lipids react to the same extent with ethyl acetimidate in the intact cell as they do in isolated membranes; this implies that the isolation does not load to major structural rearrangements.