Singh I, Lazo O, Dhaunsi G S, Contreras M
Department of Pediatrics, Medical University of South Carolina, Charleston 29425.
J Biol Chem. 1992 Jul 5;267(19):13306-13.
The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.
过氧化物酶体膜中棕榈酰辅酶A连接酶（位于细胞质表面）和二十四烷酰辅酶A连接酶（位于腔面）的不同拓扑结构表明，这些脂肪酸可能以不同形式穿过过氧化物酶体膜进行转运（拉佐，O.，孔特雷拉斯，M.，和辛格，I.（1990年）《生物化学》29卷，39�1 - 3986页），这种差异转运可能是X - 肾上腺脑白质营养不良（X - ALD）中二十四烷酸氧化缺陷的原因（辛格，I.，莫泽，A.B.，戈德菲舍尔，S.，和莫泽，H.W.（1984年）《美国国家科学院院刊》81卷，4203 - 4207页）。为了确定这些脂肪酸穿过过氧化物酶体膜的转运机制及其对X - ALD中二十四烷酸代谢的可能影响，我们研究了从大鼠肝脏分离的过氧化物酶体以及从X - ALD和对照成纤维细胞分离的过氧化物酶体中棕榈酸和二十四烷酸转运的辅助因子和能量需求。在脂肪酸活化条件下，棕榈酰辅酶A（87.6±6.3 nmol/h/mg蛋白质）和棕榈酸的转运速率相似（83.4±5.1 nmol/h/mg蛋白质），当从测定培养基中去除ATP和/或辅酶A，或用α，β - 亚甲基腺苷 - 5'-三磷酸（AMPCPOP）和/或去硫辅酶A - 琼脂糖替代时，棕榈酸的转运缺乏（为棕榈酰辅酶A转运的4%），这清楚地表明棕榈酸的转运需要过氧化物酶体细胞质表面的棕榈酰辅酶A连接酶预先合成棕榈酰辅酶A。与二十四烷酰辅酶A（0.41±0.11 nmol/h/mg蛋白质）相比，二十四烷酸的转运速率高10倍（5.3±0.6 nmol/h/mg蛋白质），并且当去除ATP和/或辅酶A，或用AMPCPOP或去硫辅酶A - 琼脂糖替代时，二十四烷酸的转运不受抑制，这表明二十四烷酸本身穿过过氧化物酶体膜进行转运。此外，去除ATP或用AMPOPCP（一种不可水解的底物）替代没有影响，这表明棕榈酰辅酶A和二十四烷酸穿过过氧化物酶体膜的转运不需要能量。在来自X - ALD的过氧化物酶体中，棕榈酸的转运、活化和氧化是正常的。与二十四烷酸正常转运到来自X - ALD的过氧化物酶体相比，二十四烷酰辅酶A连接酶缺乏（为对照的13%）以及二十四烷酸氧化缺乏（为对照的10%）清楚地表明，X - ALD中极长链脂肪酸（大于C22）的特征性积累是由于过氧化物酶体二十四烷酰辅酶A连接酶活性缺乏。
