Research for the relationship of hereditary sideroblastic anemia with enzyme complex formation in mitochondria

遗传性铁粒幼细胞贫血与线粒体酶复合物形成关系的研究

基本信息

批准号：
12670129
负责人：
FURUYAMA Kazumichi
金额：
$ 2.18万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. To examine whether Erythroid specific 5-aminolevulinate synthase (ALAS-E) and ATP-specific succinyl CoA synthethase (A-SCS) form functional enzyme complex in vitro, we have expressed recombinant proteins of components of each enzyme (ALAS-E, SCS-α, SCS-βA and SCS-βG) using Baclo-virus based expression system. In this experiment, we have successfully purified enzymatically active ALAS-E recombinant protein, however, enzymatic activity of SCS could not be detected in any combination of purified component (SCS-α and SCS-βA = A-SCS, or SCS-α and SCS-βG = G-SCS). These results suggest that phosphorilation or glycosilation SCS might be needed for enzymatically active SCS, which specifically occur in only mammalian cells. Alternatively, mammalian SCS may request specific co-factor(s) for its catalytic activity. If such co-factor(s) exist, decrease amount of the co-factor may cause sideroblastic anemia.2. To determine the specific region for interaction of ALAS-E and SCS-βA, several deletion mutants of ALAS-E protein were made. Then, the interaction of such mutant ALAS-E proteins and SCS-βA protein were determined using yeast two hybrid system. As a results, 147 amino acid deletion of N-terminal or 50 amino acid deletion of C-terminal of ALAS-E disrupt the interaction of ALAS-E and SCS-βA protein. Since these deletion mutant of ALAS-E results the disruption of homo-dimer formation of ALAS-E, homodimer formation of ALAS-E might be important for enzyme complex formation of ALAS-E and A-SCS in mitochondria.

1. 为了检查红细胞特异性5-氨基乙酰丙酸合酶（ALAS-E）和ATP特异性琥珀酰辅酶A合成酶（A-SCS）是否在体外形成功能性酶复合物，我们表达了每种酶（ALAS-E，使用基于杆状病毒的表达系统的SCS-α、SCS-βA和SCS-βG)在本实验中，我们成功地纯化了酶活性。然而，ALAS-E 重组蛋白在纯化组分的任何组合中都无法检测到 SCS 的酶活性（SCS-α 和 SCS-βA = A-SCS，或 SCS-α 和 SCS-βG = G-SCS）。表明具有酶活性的 SCS 可能需要磷酸化或糖基化 SCS，这种酶活性仅发生在哺乳动物细胞中；或者，哺乳动物 SCS 可能需要特定的辅助因子。如果存在这样的辅助因子，则辅助因子的量减少可能导致铁粒幼细胞性贫血。2．为了确定ALAS-E和SCS-βA相互作用的特定区域，几个ALAS-E的缺失突变体。然后，使用酵母二杂交系统测定了这种突变体ALAS-E蛋白和SCS-βA蛋白的相互作用，结果是N端147个氨基酸缺失或N端50个氨基酸缺失。 ALAS-E 的 C 末端破坏 ALAS-E 和 SCS-βA 蛋白的相互作用，因为 ALAS-E 的这些缺失突变体导致 ALAS-E 同二聚体形成的破坏，因此 ALAS-E 同二聚体的形成可能很重要。用于线粒体中 ALAS-E 和 A-SCS 的酶复合物形成。