Schepers L, Casteels M, Vamecq J, Parmentier G, Van Veldhoven P P, Mannaerts G P
Afdeling Farmakologie, Katholieke Universiteit Leuven, Belgium.
J Biol Chem. 1988 Feb 25;263(6):2724-31.
Rat liver and kidney homogenates, fortified with the appropriate cofactors, produced H2O2 when incubated with prostaglandin (PG) E2 or its CoA ester (PGE2-CoA), indicating that PGE2-CoA served as substrate for acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation. PGE2-CoA oxidase was stimulated to the same extent as palmitoyl-CoA oxidase by treatment of rats with the peroxisome proliferator clofibrate. Subcellular fractionation confirmed that both oxidase activities were located in peroxisomes. When highly purified peroxisomes were incubated with [1-14C]PGE2, radioactive oxidation products were released, demonstrating that peroxisomes were capable of catalyzing the complete beta-oxidation sequence. However, PGE2 beta-oxidizing activity was expressed only when isolated microsomes were added to the purified peroxisomes, indicating that PGE2-CoA synthetase is located in the endoplasmic reticulum. Cofactor requirements for peroxisomal [1-14C]PGE2 and [1-14C]palmitate oxidation were identical, and oxidation was not inhibited by cyanide. [1-14C]PGE2 was also beta-oxidized by highly purified mitochondrial fractions, devoid of peroxisomes, when microsomes were added. Mitochondrial [1-14C]PGE2 and [1-14C]palmitate oxidation was CoA- and ATP-dependent and inhibited by cyanide. Palmitate oxidation was carnitine-dependent, but PGE2 oxidation was not. Acyl-CoA dehydrogenase activity, but not carnitine acyltransferase activity, was detected with PGE2-CoA as substrate. [1-14C]PGE2 oxidation in whole liver homogenates was only slightly inhibited by cyanide, indicating that peroxisomes oxidized the major portion of PGE2. The concentrations of PGE2 employed in these experiments exceeded the in vivo concentrations by several orders of magnitude. Therefore, we suggest that the urinary PG metabolite excretion be measured in patients with hereditary diseases in which peroxisomal or mitochondrial beta-oxidation is not functioning in order to clarify the role of each organelle in PG oxidation in vivo.
用适当的辅因子强化的大鼠肝脏和肾脏匀浆，在与前列腺素（PG）E2或其辅酶A酯（PGE2 - CoA）一起孵育时会产生过氧化氢，这表明PGE2 - CoA作为过氧化物酶体β氧化的第一种酶——酰基辅酶A氧化酶的底物。用过氧化物酶体增殖剂氯贝丁酯处理大鼠后，PGE2 - CoA氧化酶受到的刺激程度与棕榈酰辅酶A氧化酶相同。亚细胞分级分离证实这两种氧化酶活性都位于过氧化物酶体中。当用[1 - 14C]PGE2孵育高度纯化的过氧化物酶体时，会释放出放射性氧化产物，这表明过氧化物酶体能够催化完整的β氧化序列。然而，只有当将分离的微粒体添加到纯化的过氧化物酶体中时，PGE2的β氧化活性才会表达，这表明PGE2 - CoA合成酶位于内质网中。过氧化物酶体对[1 - 14C]PGE2和[1 - 14C]棕榈酸氧化的辅因子需求是相同的，并且氧化不受氰化物抑制。当添加微粒体时，不含过氧化物酶体的高度纯化的线粒体部分也能将[1 - 14C]PGE2进行β氧化。线粒体对[1 - 14C]PGE2和[1 - 14C]棕榈酸的氧化是辅酶A和ATP依赖性的，并受氰化物抑制。棕榈酸氧化是肉碱依赖性的，但PGE2氧化不是。以PGE2 - CoA为底物时可检测到酰基辅酶A脱氢酶活性，但未检测到肉碱酰基转移酶活性。全肝匀浆中[1 - 14C]PGE2的氧化仅受到氰化物的轻微抑制，这表明过氧化物酶体氧化了大部分的PGE2。这些实验中使用的PGE2浓度比体内浓度高出几个数量级。因此，我们建议对过氧化物酶体或线粒体β氧化功能异常的遗传性疾病患者测量尿中PG代谢产物的排泄情况，以阐明每个细胞器在体内PG氧化中的作用。
