Axén E, Postlind H, Sjöberg H, Wikvall K
Department of Pharmaceutical Biosciences, University of Uppsala, Sweden.
Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10014-8. doi: 10.1073/pnas.91.21.10014.
A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig liver mitochondria. It also catalyzed 27-hydroxylation of 25-hydroxyvitamin D3 and 25-hydroxylation of vitamin D3. The ratio between the 1 alpha-, 27-, and 25-hydroxylase activities remained essentially constant during the purification. Substrates for sterol 27-hydroxylase CYP27 inhibited and a monoclonal antibody raised against CYP27 immunoprecipitated the 1 alpha-, 27-, and 25-hydroxylase activities. Apparently homogeneous preparations of CYP27 from pig and rabbit liver mitochondria catalyzed 1 alpha-hydroxylation. Human liver mitochondrial CYP27 was expressed from its cDNA in Escherichia coli. The nucleotide sequence encoding the N terminus of CYP27 was modified in the first eight codons to achieve expression in E. coli. The purified recombinant-expressed CYP27 reconstituted with the electron-transferring system of adrenal mitochondria catalyzed 1 alpha-hydroxylation of 25-hydroxyvitamin D3. Expression of unmodified CYP27 cDNA in simian COS cells confirmed the 1 alpha-hydroxylase activity toward 25-hydroxyvitamin D3.
一种催化25-羟基维生素D3 1α-羟化的细胞色素P450从猪肝线粒体中纯化得到。它还催化25-羟基维生素D3的27-羟化以及维生素D3的25-羟化。在纯化过程中，1α-、27-和25-羟化酶活性之间的比例基本保持恒定。固醇27-羟化酶CYP27的底物具有抑制作用，针对CYP27产生的单克隆抗体可免疫沉淀1α-、27-和25-羟化酶活性。从猪肝线粒体和兔肝线粒体中获得的明显均一的CYP27制剂催化1α-羟化反应。人肝线粒体CYP27通过其cDNA在大肠杆菌中表达。编码CYP27 N端的核苷酸序列在前八个密码子中进行了修饰，以实现在大肠杆菌中的表达。用肾上腺线粒体的电子传递系统重构的纯化重组表达CYP27催化25-羟基维生素D3的1α-羟化反应。未修饰的CYP27 cDNA在猴COS细胞中的表达证实了其对25-羟基维生素D3的1α-羟化酶活性。
