CHARACTERIZATION OF MAMMALIAN FERROCHELATASE

本提案的目的是检测 血红素的生物合成途径--铁络合酶(Protoheme Ferroyase，E.C. 4.99.1.1.)，以确定关于 来自健康动物组织的酶。牛肝将成为 自从遗传原卟啉在牛身上被描述以来，这种酶就已经被描述出来了。一次 从正常动物身上获得这种酶的信息，然后它就会 有可能检测患有脑炎的动物的铁络合酶 这样我们就可以了解它的分子基础 疾病。这里概述的实验方法包括检查 铁络合酶在膜中的定位，可能性的定义 蛋白质-脂质和蛋白质-蛋白质相互作用及其催化研究 酶的功能。这些数据将主要来自化学品 纯化酶的修饰研究及酶促活性的检测 重组脂质体系统中的相互作用和通过使用 卟啉底物类似物和抑制剂。这样做的最终目的是 实验室正在对三种末端膜进行生化表征 这一途径的相关酶，并将它们重组为脂质体 当它们在原位时，它们在膜上以矢量方式组织 在线粒体中。该系统的研究首先在正常动物和 然后在斑点动物身上，以及与临床医生的合作研究 在这个领域，可能会导致对生物化学和分子生物学的理解 原卟啉症和杂色卟啉症。

The objective of the present proposal is to examine the terminal enzyme in the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, E.C. 4.99.1.1.), to determine structural and catalytic information about the enzyme from healthy animal tissue. Bovine liver will be the source of enzyme since a genetic protoporphyria has been described in cattle. Once information is obtained about the enzyme from normal animals, then it will be possible to examine ferrochelatase from animals suffering from protoporphyria so that we may then understand the molecular basis of that disease. The experimental approach outlined herein involves examination of the orientation of ferrochelatase in membranes, definition of possible protein-lipid and protein-protein interactions and study of the catalytic functioning of the enzyme. The data will come largely from chemical modification studies on the purified enzyme as well as examination of interactions in reconstituted liposomal systems and through the use of porphyrin substrate analogs and inhibitors. The ultimate goal of this laboratory is to biochemically characterize the three terminal membrane associated enzymes of this pathway and reconstitute them into liposomes so that they are vectorally organized across the membrane as they are in situ in the mitochondria. The study of the system first in normal animals and then in porphyric animals, along with collaborative studies with clinicians in he field, may lead to the biochemical and molecular understanding of protoporphyria and variegate porphyria.