Palosaari P M, Kilponen J M, Sormunen R T, Hassinen I E, Hiltunen J K
Department of Medical Biochemistry, University of Oulu, Finland.
J Biol Chem. 1990 Feb 25;265(6):3347-53.
Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.
Δ3,Δ2-烯酰辅酶A异构酶（EC 5.3.3.8）是脂肪酸β-氧化过程中奇数位置双键代谢所必需的辅助酶，在正常大鼠和氯贝特处理的大鼠的心脏和肝脏中进行了研究。肝脏过氧化物酶体和线粒体同工酶可通过染料配体色谱法分离。线粒体同工酶进一步纯化至表观均一性。还从心肌（过氧化物酶体含量低的组织）中纯化出一种异构酶。这些酶是二聚体碱性蛋白（pI 9.5），亚基分子量为30,000。顺式和反式烯酰辅酶A都是所研究的肝脏酶的底物。C6-、C10-和C12-反式-3-烯酰底物的速度比为9:2.5:1。通过免疫电子显微镜发现，所选纯化的酶蛋白在肝脏和心脏中均为线粒体来源。色谱证据、免疫电子显微镜和免疫印迹表明，在肝脏而非心脏中，该酶在氯贝特处理期间诱导增加了1个数量级。针对大鼠异构酶的抗体在牛、猪肝和心脏以及人胎盘中检测到交叉反应蛋白。估计的亚基大小因物种而异，牛肝脏和心脏中为31,000，猪肝脏和心脏中为29,000，人胎盘中为30,000。这些数据与Δ3,Δ2-烯酰辅酶A异构酶的双重定位概念一致。通过免疫化学验证了其中一种同工酶的线粒体起源及其对氯贝特的组织特异性诱导，并通过蛋白质的色谱行为揭示了过氧化物酶体同工酶的身份。
