Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase.
Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase.
复制标题
大鼠肝微粒体维生素 D3-25-羟化酶可溶性成分的分离和重建。
DOI：
10.1016/0003-9861(80)90210-6
复制
发表时间：
1980
影响因子：
3.9
通讯作者：
Deluca,HF
中科院分区：
文献类型：
--
作者：
Yoon,PS;Deluca,HF
从大鼠肝微粒体中分离的维生素D3-25-羟化酶的可溶性组分，已知其在体外催化维生素D3的C-25氧化，已被分离成两个亚微粒体组分，富含去污剂增溶的NADPH-细胞色素还原酶和细胞色素P-450或P-448。通过用胆酸溶解，然后用非离子洗涤剂Emulgen 911处理，获得含P-450血红蛋白的部分，得到最终制剂，其特定含量为7.25 nmol/mg微粒体蛋白。通过用脱氧胆酸盐增溶和在DEAE-纤维素上层析，从细胞色素b5 orP-450中分离出减少的三磷酸吡啶核苷酸依赖性细胞色素P-450还原酶活性，通过其减少人工电子受体细胞色素c的能力来检测。还原酶组分的米氏常数Km（NADPH）= 3.14μM，Km（NADH）= 31.25μM，Km（cytc）= 12.34μM。NADPH-细胞色素还原酶活性对NADP的NADPH可逆抑制敏感，但对鱼藤酮或氰化物不敏感。当分离的组分在无氧条件下在存在NADPH生成系统和一氧化碳的情况下孵育时，通过还原的一氧化碳与还原的差异光谱在420和450 nm处出现的特征吸光度来测量P-450血红素蛋白的酶还原。此外，当可溶性亚微粒体组分用过量还原的三磷酸吡啶核苷酸、3 H标记的维生素D3和可溶性胞质上清液重建时，完全维生素D3-25-羟化酶活性以高达7.68 pmol/h/mg蛋白的速率恢复，在血红素蛋白浓度限制净产物形成速率的条件下，细胞色素P-450的表观周转数为1.16 - 1.20。这些结果有力地支持了以下假设：大鼠肝微粒体混合功能氧化酶，维生素D3-25-羟化酶，由至少两个膜结合蛋白组分，NADPH-细胞色素还原酶和细胞色素P-450末端氧化酶组成，用于催化维生素D3转化为25-羟基维生素D3。
Solubilized components of the vitamin D3-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D3in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochromecreductase and cytochromeP-450 orP-448. TheP-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochromeP-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochromec, was isolated free of cytochromesb5orP-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants:Km(NADPH)= 3.14μM,Km(NADH)= 31.25μM, andKm(cytc)= 12.34μM. The NADPH-cytochromecreductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of theP-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide,3H-labeled vitamin D3, and soluble cytosolic supernatant, full vitamin D3-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochromeP-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D3-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochromecreductase and a cytochromeP-450 terminal oxidase, for the catalytic conversion of vitamin D3to 25-hydroxyvitamin D3.