Lazo O, Contreras M, Hashmi M, Stanley W, Irazu C, Singh I
Department of Pediatrics, Medical University of South Carolina, Charleston 29425.
Proc Natl Acad Sci U S A. 1988 Oct;85(20):7647-51. doi: 10.1073/pnas.85.20.7647.
We previously reported that in childhood adrenoleukodystrophy (C-ALD) and adrenomyeloneuropathy (AMN), the peroxisomal beta-oxidation system for very long chain (greater than C22) fatty acids is defective. To further define the defect in these two forms of X chromosome-linked ALD, we examined the oxidation of [1-14C]lignoceric acid (n-tetracosanoic acid, C24:0) and [1-14C]lignoceroyl-CoA (substrates for the first and second steps of beta-oxidation, respectively). The oxidation rates of lignoceric acid in C-ALD and AMN were 43% and 36% of control values, respectively, whereas the oxidation rate of lignoceroyl-CoA was 109% (C-ALD) and 106% (AMN) of control values, respectively. On the other hand, the oxidation rates of palmitic acid (n-hexadecanoic acid) and palmitoyl-CoA in C-ALD and AMN were similar to the control values. These results suggest that lignoceroyl-CoA ligase activity may be impaired in C-ALD and AMN. To identify the specific enzymatic deficiency and its subcellular localization in C-ALD and AMN, we established a modified procedure for the subcellular fractionation of cultured skin fibroblasts. Determination of acyl-CoA ligase activities provided direct evidence that lignoceroyl-CoA ligase is deficient in peroxisomes while it is normal in mitochondrial and microsomes. Moreover, the normal oxidation of lignoceroyl-CoA as compared with the deficient oxidation of lignoceric acid in isolated peroxisomes also supports the conclusion that peroxisomal lignoceroyl-CoA ligase is impaired in both C-ALD and AMN. Palmitoyl-Coa ligase activity was found to be normal in peroxisomes as well as in mitochondria and microsomes. This normal peroxisomal palmitoyl-CoA ligase activity as compared with the deficient activity of lignoceroyl-CoA ligase in C-ALD and AMN suggests the presence of two separate acyl-CoA ligases for palmitic and lignoceric acids in peroxisomes. These data clearly demonstrate that the pathognomonic accumulation of very long chain fatty acids in C-ALD and AMN is due to a deficiency of peroxisomal very long chain (lignoceric acid) acyl-CoA ligase.
我们之前报道过，在儿童肾上腺脑白质营养不良（C-ALD）和肾上腺脊髓神经病（AMN）中，过氧化物酶体中用于极长链（大于C22）脂肪酸的β-氧化系统存在缺陷。为了进一步明确这两种X染色体连锁的ALD形式中的缺陷，我们检测了[1-14C]二十四烷酸（正二十四烷酸，C24:0）和[1-14C]二十四烷酰辅酶A（分别为β-氧化第一步和第二步的底物）的氧化情况。C-ALD和AMN中二十四烷酸的氧化速率分别为对照值的43%和36%，而二十四烷酰辅酶A的氧化速率分别为对照值的109%（C-ALD）和106%（AMN）。另一方面，C-ALD和AMN中棕榈酸（正十六烷酸）和棕榈酰辅酶A的氧化速率与对照值相似。这些结果表明，C-ALD和AMN中二十四烷酰辅酶A连接酶的活性可能受损。为了确定C-ALD和AMN中具体的酶缺陷及其亚细胞定位，我们建立了一种改良的培养皮肤成纤维细胞亚细胞分级分离方法。酰基辅酶A连接酶活性的测定提供了直接证据，表明二十四烷酰辅酶A连接酶在过氧化物酶体中缺乏，而在线粒体和微粒体中正常。此外，与分离的过氧化物酶体中二十四烷酸氧化缺陷相比，二十四烷酰辅酶A的正常氧化也支持了C-ALD和AMN中过氧化物酶体二十四烷酰辅酶A连接酶受损的结论。发现棕榈酰辅酶A连接酶活性在过氧化物酶体以及线粒体和微粒体中均正常。与C-ALD和AMN中二十四烷酰辅酶A连接酶活性缺陷相比，这种过氧化物酶体中正常的棕榈酰辅酶A连接酶活性表明过氧化物酶体中存在两种分别用于棕榈酸和二十四烷酸的酰基辅酶A连接酶。这些数据清楚地表明，C-ALD和AMN中极长链脂肪酸的特征性蓄积是由于过氧化物酶体极长链（二十四烷酸）酰基辅酶A连接酶缺乏所致。
