PURIFICATION OF PEROXISOMES AND SUBCELLULAR-DISTRIBUTION OF ENZYME-ACTIVITIES FOR ACTIVATION AND OXIDATION OF VERY-LONG-CHAIN FATTY-ACIDS IN RAT-BRAIN
PURIFICATION OF PEROXISOMES AND SUBCELLULAR-DISTRIBUTION OF ENZYME-ACTIVITIES FOR ACTIVATION AND OXIDATION OF VERY-LONG-CHAIN FATTY-ACIDS IN RAT-BRAIN
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DOI：
10.1016/0005-2760(93)90174-8
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发表时间：
1993-09-29
期刊：
影响因子：
--
通讯作者：
KREMSER, K
中科院分区：
文献类型：
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作者：
SINGH, I;LAZO, O;KREMSER, K
大脑含有大量的极长链 (VLC) 脂肪酸 (> C22)。由于肝脏和皮肤成纤维细胞的线粒体缺乏木蜡酰辅酶A连接酶，因此在肝脏和皮肤成纤维细胞中，脂肪酸仅在过氧化物酶体中被氧化。 Poulos 及其同事的研究结果 [9] 表明，与肝脏和培养的皮肤成纤维细胞相反，脑线粒体含有木蜡酰辅酶 A 连接酶，可以氧化木蜡酸。本研究的目的是开发一种从大脑中分离出更高纯度的亚细胞细胞器的方法，并更好地了解大脑中 VLC 脂肪酸氧化的亚细胞定位。在来自大鼠脑的过氧化物酶体、线粒体、微粒体和髓磷脂级分以及从大鼠肝脏纯化的过氧化物酶体、线粒体和微粒体中测定了棕榈酸和木蜡酸活化和氧化的酶活性。与肝脏一样，脑微粒体和过氧化物酶体中的木质素辅酶 A 连接酶活性约为比线粒体高 9 倍。除了棕榈酰辅酶A连接酶之外，针对棕榈酰辅酶A连接酶的抗体抑制残留的线粒体木脂酰辅酶A连接酶活性，这意味着线粒体中的木脂酰辅酶A连接酶活性源自棕榈酰辅酶A连接酶。因此，过氧化物酶体中木蜡酸的氧化速度是线粒体中的 7 倍。当补充来自微粒体或髓磷脂的木蜡酰辅酶A连接酶活性时，线粒体能够有效地氧化木蜡酸。这些结果表明，在脑中，木蜡酸在过氧化物酶体中被氧化，木蜡酰辅酶A连接酶活性位于过氧化物酶体和微粒体中，但不在线粒体中。过氧化物酶体和微粒体含有木蜡酰辅酶 A 和棕榈酰辅酶 A 连接酶。与过氧化物酶体和微粒体类似，针对棕榈酰辅酶A连接酶的抗体仅抑制髓磷脂中的棕榈酰辅酶A连接酶活性，但不抑制木质素酰辅酶A连接酶活性。这些结果表明，除了棕榈酰辅酶A连接酶之外，髓磷脂还含有二十四酰辅酶A连接酶。
Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22). Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes. Findings by Poulos and associates [9] suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid. The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain. The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver. Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx. 9 times higher than in mitochondria. In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase. Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria. Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin. These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria. Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases. Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity. These results suggest that in addition to palmitoyl-CoA ligase, myelin also contains lignoceroyl-CoA ligase.