Wanders R J, van Roermund C W, van Wijland M J, Schutgens R B, van den Bosch H, Schram A W, Tager J M
Department of Pediatrics, University Hospital Amsterdam, The Netherlands.
Biochem Biophys Res Commun. 1988 Jun 16;153(2):618-24. doi: 10.1016/s0006-291x(88)81140-9.
A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].
已开发出一种从培养的皮肤成纤维细胞中制备富含过氧化物酶体且不含微粒体和线粒体的组分的方法。该方法包括对核后上清液进行差速离心，随后在不连续的甲泛葡胺梯度上进行密度梯度离心。随后在来自对照受试者和患有X连锁肾上腺脑白质营养不良症患者的培养皮肤成纤维细胞制备的核后上清液和富含过氧化物酶体的组分中测量二十六烷酰辅酶A合成酶的活性。虽然X连锁肾上腺脑白质营养不良症成纤维细胞核后上清液中的二十六烷酰辅酶A合成酶活性仅略有下降（为对照的77.8±4.4%（n = 15）），但发现从突变成纤维细胞中分离的过氧化物酶体组分中的酶活性下降更为明显（为对照的19.6±6.7%（n = 5））。这直接证明了X连锁肾上腺脑白质营养不良症的缺陷在于过氧化物酶体激活极长链脂肪酸的能力不足，正如Hashmi等人首先提出的那样[Hashmi, M., Stanley, W.和Singh, I. (1986) FEBS Lett. 86, 247 - 250]。
