Loading of iron into recombinant rat liver ferritin heteropolymers by ceruloplasmin.
Loading of iron into recombinant rat liver ferritin heteropolymers by ceruloplasmin.
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通过铜蓝蛋白将铁负载到重组大鼠肝铁蛋白杂聚物中。
DOI：
10.1006/abbi.1997.9967
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发表时间：
1997
影响因子：
3.9
通讯作者：
S. Aust
中科院分区：
文献类型：
--
作者：
S. Juan;J. Guo;S. Aust
我们之前曾报道过，铜蓝蛋白掺入铁需要铁蛋白重链（J. - H. Guo，M. Abedi和S. D. Aust（1996）Arch. Biochem. Biophys. 335（1））。本研究的目的是确定铜蓝蛋白与铁蛋白相互作用所需的重链数量，从而发生铁负载。将编码大鼠肝铁蛋白重链和轻链的cDNA序列分别克隆到杆状病毒转移载体pA-cUW 51中，并通过同源重组将其整合到苜蓿银纹夜蛾核型多角体病毒基因组中。重组病毒感染昆虫细胞后，两条铁蛋白链均得到表达，并组装成两种异聚体，经DEAE-Sepharose层析分离。组成两种杂聚物的重链（H）和轻链（L）的百分比（在SDS-PAGE上解析链后通过凝胶扫描测定）相当于1 H和23 L链以及2 H和22 L链。使用1摩尔的大鼠血浆铜蓝蛋白每摩尔的H链中的两个杂聚物的铁负载的最大程度进行了观察。铁掺入的程度降低与额外的血浆铜蓝蛋白。铁掺入到大鼠肝铁蛋白，发现含有10个H链，增加血浆铜蓝蛋白铁蛋白的摩尔比增加到4：1，并保持不变，高达8：1。铁加载到马脾铁蛋白，发现有一个H链，出现类似的重组铁蛋白，只有一个H链。因此，我们建议，最佳的铜蓝蛋白与铁蛋白的摩尔比取决于H链的数目，使铁的最大掺入到铁蛋白的铁蛋白分子。这些结果还表明，铁负载通道包含在一个单一的H链亚基。
We have reported previously that the heavy chain of ferritin is required for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for ceruloplasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferritin were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infections Autographa californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the infection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light (L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H chain in the two heteropolymers. The extent of iron incorporation decreased with additional ceruloplasmin. Iron incorporation into rat liver ferritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up to 8:1. Iron loading into horse spleen ferritin, found to have one H chain, appeared similar to that for recombinant ferritin, having only one H chain. Therefore, we propose that the optimal molar ratio of ceruloplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin. These results also suggest that the iron loading channel is contained within a single H chain subunit.
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影响因子：
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