Singh I, Kishimoto Y
J Neurochem. 1981 Aug;37(2):388-91. doi: 10.1111/j.1471-4159.1981.tb00467.x.
The particulate fraction, heat-labile factor, heat-stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (alpha-hydroxylignoceric acid). The heat-labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD-Agarose affinity chromatography. The specific activity of the heat-labile factor was increased 105-fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS-polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat-labile factor was incubated with 1 mM-N-ethylmaleimide. This inhibition was prevented by preincubating the heat-labile factor with 1 mM-NADH. These observations indicate that the heat-labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.
颗粒部分、热不稳定因子、热稳定因子和NADPH对于木蜡酸（二十四烷酸）转化为脑苷脂酸（α-羟基木蜡酸）至关重要。热不稳定因子从小牛小脑提取，并通过硫酸铵沉淀、羟基磷灰石色谱、等电聚焦和NAD-琼脂糖亲和色谱四个步骤进行部分纯化。在最后三个步骤中，热不稳定因子的比活性提高了105倍，活性产率为37%。用考马斯亮蓝染色的SDS-聚丙烯酰胺凝胶电泳检测该制剂时，可见一条主要条带和几条次要条带。主要条带对应分子量为32700的蛋白质，次要条带对应分子量为62000和67000的蛋白质。当热不稳定因子与1 mM N-乙基马来酰亚胺孵育时，活性丧失。通过将热不稳定因子与1 mM NADH预孵育可防止这种抑制作用。这些观察结果表明，热不稳定因子含有一个对活性至关重要的巯基，并且它位于吡啶核苷酸结合位点处或附近。
