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Structural analysis of regulatory factors of complement on erythrocyte membranes

红细胞膜上补体调节因子的结构分析

基本信息

批准号：
62580132
负责人：
TOMITA Motowo
金额：
$ 1.41万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

Erythrocytes are hemolyzed with hetrologous complement, but not with homologous one; this evidence is called homologous restriction of complement. It has been suggested that an unidentified membrane factor is responsible for the restriction.I tried to identify the factor. DAF, decay-accerelating factor of C3,C5-convertase, seemed to be the first candidate at the starting stage of this project, because DAF regulates the complement activation on erythrocytes. DAF was isolated from rabbit erythrocytes by an improved method applied. Both of rabbit and human DAFs showed only a weak homologous restriction. Thus, I concluded that DAF is not a major factor responsible for the restriction.HRF, an inhibitor of membrane attack complex(MAC)-formation on human erythrocytes reported in 1986, was chosen for the second candidate. HRF, however, could not be isolated by the method described in the original paper, because the assay method was unreliable. I developed a new reliable method for detecting the inhibitor of MAC-formation, and isolated a new factor of a molecular weight 18Kd, designated MACIF. MACIF showed a potent inhibitory activity to human complement, but not to guinea pig one indicating a likely candidate responsible for the restriction. I succeeded in cDNA cloning of MACIF mRNA and determined the complete amino acid sequence of MACIF.

红细胞被异源补体溶血，但不被同源补体溶血，这种现象称为补体的同源限制。有人认为是一种未知的膜因子造成了这种限制，我试图确定这种因子。C3，C5-转化酶的衰变加速相关因子，在本项目的开始阶段似乎是第一个候选者，因为C3，C5-转化酶调节红细胞上的补体活化。采用改良的方法从家兔红细胞中分离纯化β-内酰胺酶。兔和人的DAF仅显示出弱的同源限制。因此，我的结论是，HRF不是一个主要的因素负责的限制。HRF，膜攻击复合物（MAC）的形成抑制剂对人红细胞在1986年报道，被选为第二个候选人。然而，HRF不能被分离的方法中描述的原始文件，因为该测定方法是不可靠的。本研究建立了一种新的检测MAC-形成抑制因子的可靠方法，并分离到一个分子量为18 Kd的新因子，命名为MACIF。MACIF对人补体显示出有效的抑制活性，但对豚鼠补体没有抑制活性，这表明可能是负责限制的候选物。我成功地克隆了MACIF mRNA的cDNA，并测定了MACIF的完整氨基酸序列。