Horie S, Suzuki T, Suga T
Department of Clinical Biochemistry, Tokyo College of Pharmacy, Japan.
Arch Biochem Biophys. 1989 Oct;274(1):64-73. doi: 10.1016/0003-9861(89)90415-3.
The acetyl-CoA-dependent elongation of medium-chain acyl-CoA in the presence of pyridine nucleotide was studied in rat liver. The activity was increased by the administration of peroxisome proliferators, clofibrate and di-(2-ethylhexyl)phthalate, and the change was more remarkable in peroxisomes than in mitochondria. Addition of 0.01% Triton X-100 to the incubation mixture caused an increase in the mitochondrial activity, whereas the peroxisomal activity did not increase significantly. The pH optimum for the peroxisomal activity was in the range of pH 6.5-7.0 and that for the mitochondrial activity was pH 7.5-8.0. The specificities of primer chain length in both organelles were almost the same, and octanoyl-CoA was the preferred substrate. Peroxisomal activity was completely inhibited by the addition of 1 mM N-ethylmaleimide or 1 mM p-hydroxymercuribenzoic acid, while the activity did not change on the addition of 1 mM KCN or an antibody to acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system. The activity of enoyl-CoA reductase, which catalyzes the last step of the elongation system, was also detected in peroxisomes, although the main activity was localized in microsomes. When the liver peroxisomal fraction of clofibrate-treated rats was incubated with a mixture of octanoyl-CoA, acetyl-CoA, NADH, NADPH, and Triton X-100 in a buffer system, dodecanoyl-CoA was detected as the main product by radio-gas chromatography. On the other hand, the elongation activity was decreased greatly by the addition of NAD+ into the mixture. These results indicate that (i) peroxisomes have activity to elongate medium chain acyl-CoA; (ii) the peroxisomal elongation system may consist of the reverse reaction of the beta-oxidation system except for the last step, which is catalyzed by enoyl-CoA reductase; and (iii) the peroxisomal elongation system is less active than the beta-oxidation system under physiological conditions.
在大鼠肝脏中研究了在吡啶核苷酸存在下，乙酰辅酶A依赖的中链酰基辅酶A的延长反应。给予过氧化物酶体增殖剂氯贝丁酯和邻苯二甲酸二（2-乙基己基）酯后，该活性增强，且过氧化物酶体中的变化比线粒体中更显著。向孵育混合物中添加0.01%的 Triton X-100会导致线粒体活性增加，而过氧化物酶体活性没有显著增加。过氧化物酶体活性的最适pH在6.5 - 7.0范围内，线粒体活性的最适pH为7.5 - 8.0。两种细胞器中引物链长度的特异性几乎相同，辛酰辅酶A是首选底物。添加1 mM N-乙基马来酰亚胺或1 mM对羟基汞苯甲酸可完全抑制过氧化物酶体活性，而添加1 mM KCN或抗酰基辅酶A氧化酶（过氧化物酶体β氧化系统的第一种酶）的抗体时，活性没有变化。尽管主要活性定位于微粒体中，但在过氧化物酶体中也检测到了催化延长系统最后一步的烯酰辅酶A还原酶的活性。当用氯贝丁酯处理的大鼠肝脏过氧化物酶体部分在缓冲系统中与辛酰辅酶A、乙酰辅酶A、NADH、NADPH和Triton X-100的混合物一起孵育时，通过放射性气相色谱法检测到十二烷酰辅酶A是主要产物。另一方面，向混合物中添加NAD⁺会使延长活性大大降低。这些结果表明：（i）过氧化物酶体具有延长中链酰基辅酶A的活性；（ii）过氧化物酶体延长系统可能由β氧化系统的逆反应组成，除了最后一步由烯酰辅酶A还原酶催化；（iii）在生理条件下，过氧化物酶体延长系统的活性低于β氧化系统。
