Laulhere J P, Laboure A M, Briat J F
Laboratoire de Biologie Moléculaire Végétale, Centre National de la Recherche Scientifique UA 1178, Université Joseph Fourier (Grenoble I), France.
J Biol Chem. 1989 Feb 25;264(6):3629-35.
Soluble and insoluble forms of ferritins have been purified from dry pea seeds by gel filtration. The insoluble form is called phytosiderin by analogy with animal hemosiderin. Native gel electrophoresis of these two forms have shown that the soluble one (ferritin) is homogenous in size and more compact than the insoluble one (phytosiderin) which is heterogenous in size. However, when iron is removed from these two classes of molecules (apoferritin), they have the same mobility in isopycnic centrifugations. Polyacrylamide-sodium dodecyl sulfate gel electrophoresis revealed a difference in their subunit composition: ferritin molecules are built up from a 28-kDa subunit and phytosiderin from a 26.5-kDa subunit. Partial proteolysis using a Staphylococcus aureus protease indicates a strong relationship between these two polypeptides. Intermediates between these two forms have also been characterized and are composed of both subunits in various amounts. Ferritin and phytosiderin are both able to incorporate iron in vitro into their mineral core. It is also shown that in vitro iron exchange induces ferritin degradation. This degradation is prevented by inhibitors of the Fenton cycle (iron chelates like o-phenanthroline and desferrioxamine B) and reduced by Tris, a radical scavenger. Under in vitro conditions of controlled radical damage the 28-kDa subunit is converted into the 26.5-kDa subunit. Purification of the 28-kDa subunit has allowed us to determine the NH2-terminal sequence. The NH2 extremity of the 26.5-kDa subunit is heterogenous, but the sequence of its main component is identical to the sequence of the 28-kDa subunit downstream residue Leu-21. These data indicate that the 26.5-kDa subunit is produced by radical mediated damage leading to a series of cleavages in the NH2 terminal part of the 28-kDa subunit.
通过凝胶过滤从干豌豆种子中纯化出了可溶性和不溶性两种形式的铁蛋白。不溶性形式因类似于动物血铁黄素而被称为植物铁黄素。对这两种形式进行的非变性凝胶电泳显示，可溶性形式（铁蛋白）大小均一，且比大小不均一的不溶性形式（植物铁黄素）结构更紧密。然而，当从这两类分子（脱铁铁蛋白）中去除铁时，它们在等密度离心时具有相同的迁移率。聚丙烯酰胺 - 十二烷基硫酸钠凝胶电泳揭示了它们亚基组成的差异：铁蛋白分子由28 kDa的亚基构成，而植物铁黄素由26.5 kDa的亚基构成。使用金黄色葡萄球菌蛋白酶进行部分蛋白水解表明这两种多肽之间存在密切关系。这两种形式之间的中间产物也已得到表征，并由不同数量的两种亚基组成。铁蛋白和植物铁黄素在体外均能将铁掺入其矿物核心中。还表明体外铁交换会诱导铁蛋白降解。芬顿循环抑制剂（如邻菲罗啉和去铁胺B等铁螯合剂）可防止这种降解，而自由基清除剂Tris可使其降解减少。在体外可控自由基损伤条件下，28 kDa亚基会转化为26.5 kDa亚基。对28 kDa亚基的纯化使我们能够确定其NH2末端序列。26.5 kDa亚基的NH2末端是不均一的，但其主要成分的序列与28 kDa亚基下游Leu - 21残基的序列相同。这些数据表明，26.5 kDa亚基是由自由基介导损伤产生的，导致28 kDa亚基的NH2末端部分发生一系列裂解。
