HEPATIC MICROSOMAL LIPID PEROXIDASE

这项建议的长期目标是了解 负责细胞过氧化碎裂的系统 内质网多不饱和脂肪酸对某些物质的反应 药物、化学品和不利条件。年取得的初步成果 这笔赠款的上一个资助年度(1988)(GM26548-08)表明 脂类不是简单的自氧化、自由基驱动的反应 过氧化可能，至少部分是由于一种活性的 此前未见报道的酶。对这种酶有了了解， 它的活性与药物的细胞毒性，细胞坏死， 衰老和肿瘤形成，可能导致这些最终的控制 条件。计划进行的研究包括分离、纯化和 该酶的性质，其动力学和 作用机制。初始增溶和分馏方案 将被修饰以尝试最大限度地提高活性酶的产量。尝试 将被用来稳定这种极不稳定的酶。这个 酶将以其最小分子量(SDS-PAGE)进行表征， UV/可见光光谱，可能的假体基团或金属离子含量 氨基酸末端氨基酸序列。底物的专一性 将测定酶，包括磷脂类偏好和脂肪 酰基专一性，使用重新构建的系统并可在商业上获得 纯磷脂。纯化的大鼠肝脏将产生抗体 蛋鸡或兔子体内的酶，以便a)定位酶 关于亚细胞室，b)确定组织位置 以及c)以能够分离出表达脂质的cDNA克隆 过氧化物酶蛋白。克隆将被用来推断完整的 酶的氨基酸序列，并形成用于监测的探针 脂质过氧化物酶转录活性对药物挑战的响应。这个 后者，加上抗体的使用，将有助于通过以下方式识别机制 哪些药物和化学物质会在体内引发脂质过氧化，即， 转录，或蛋白质或信使核糖核酸稳定。

The long term goal of this proposal is to gain an understanding of the system responsible for the peroxidative fragmentation of the polyunsaturated fatty acids of endoplasmic reticulum in response to certain drugs, chemicals and adverse conditions. Preliminary results obtained in the last funded year (1988) of this grant (GM26548-08) suggested that rather than a simple autooxidative, radical-driven reaction, lipid peroxidation may, at least in part, be the result of activity of a previously unreported enzyme. Gaining an understanding of this enzyme, the activity of which has been implicated in drug cytotoxicity, cell necrosis, aging and tumorigenesis, may lead to the eventual control of these conditions. Studies planned include the isolation, purification and characterization of this enzyme, and determination of its kinetics and mechanism of action. The initial solubilization and fractionation schemes will be modified to attempt to maximize yields of active enzyme. Attempts will be made to stabilize the enzyme, which is extremely labile. The enzyme will be characterized with its minimum molecular weight (SDS-PAGE), UV/visible spectrum, possible prosthetic group or metal ion content and amino terminal amino acid sequence. The substrate specificity of the enzyme will be determined, both phospholipid class preference and fatty acyl specificity, using a reconstituted system and commercially available pure phospholipids. Antibodies will be raised to the purified rat liver enzyme in either laying hens or rabbits in order to a) localize the enzyme with respect to subcellular compartment, b) determine the tissue locations of the enzyme and c) to enable isolation of cDNA clones expressing lipid peroxidase protein. The clones will be used both to deduce the complete amino acid sequence of the enzyme and to form probes with which to monitor lipid peroxidase transcription activity in response to drug challenge. The latter, plus antibody use, will aid in identification of the mechanisms by which drugs and chemicals act to trigger lipid peroxidation in vivo, i.e., transcription, or protein or mRNA stabilization.