Gustafsson J
J Biol Chem. 1975 Oct 25;250(20):8243-7.
Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.
在大鼠肝脏中，3α,7α,12α-三羟基-5β-[7β-³H]胆甾烷酸转化为3α,7α,12α,24-四羟基-5β-胆甾烷酸的过程，可由添加了100,000倍重力离心后的上清液的线粒体部分，或添加了100,000倍重力离心后的上清液和ATP的微粒体部分催化。微粒体系统比线粒体系统更具活性。对于微粒体系统，以游离的3α,7α,12α-三羟基-5β-胆甾烷酸作为底物时的反应速率，比以相应的辅酶A酯作为底物时要快得多。添加辅酶A会抑制活性。添加除ATP和辅酶A之外的其他辅因子对反应没有明显影响。100,000倍重力离心后的上清液可用通过对100,000倍重力离心后的上清液进行硫酸铵沉淀和葡聚糖凝胶柱层析得到的蛋白质部分替代。该反应不是由混合功能氧化酶催化的，因为在含¹⁸O₂的气氛中进行反应时，产物中没有¹⁸O的掺入。另一方面，氧气可能是必需的，因为在由氮气组成的气氛中进行反应时，反应几乎完全被抑制。一氧化碳不抑制该反应。当在含有重水的介质中进行反应时，产物中掺入了一个氘原子。得出的结论是，微粒体对3α,7α,12α-三羟基-5β-胆甾烷酸的24-羟基化作用涉及去饱和酶和水合酶的联合作用。水合酶催化的反应似乎具有立体特异性，因为3α,7α,12α-三羟基-5β-胆甾烷酸的24α差向异构体是主要产物。与微粒体系统不同，线粒体系统不受ATP添加的刺激，也不受辅酶A的抑制。3α,7α,12α-三羟基-5β-胆甾烷酸的辅酶A酯比游离酸更有效地进行24-羟基化。
