Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity.
Avian kidney mitochondrial hemeprotein P-4501 alpha: isolation, characterization and NADPH-ferredoxin reductase-dependent activity.
复制标题
禽肾线粒体血红素蛋白 P-4501 α：分离、表征和 NADPH-铁氧还蛋白还原酶依赖性活性。
DOI：
10.1016/0304-4165(90)90044-w
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Ghazarian,JG
中科院分区：
文献类型：
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作者：
Mandel,ML;Swartz,SJ;Ghazarian,JG
本文报道从鸡肾线粒体中分离细胞色素P-4501α。虽然凝胶渗透HPLC法得到的P-450占胆酸盐溶解血红素蛋白中P-450总量的41%，但它产生了高度纯化的混合物，通过使用羟丙基硅胶的疏水相互作用HPLC的一步应用，P-4501α可以在最终的无洗涤剂状态下纯化至均匀。纯化的P-4501α在SDS凝胶电泳中呈单一条带，表观Mr = 57000。P-4501α（Fe ~（3+））的绝对光谱在403 nm处有一个λ max。这一特性支持了我们以前报道过的P-4501α的反常高自旋血红素电子顺磁共振谱和血红素结构（Ghazarian等（1980）J.Biol.Chem.255，8275-8281; Pedersen等（1976）J.Biol.Chem.251，3933-3941）。在铁氧还蛋白依赖性NADPH-细胞色素（P-450）还原酶复合物的重建实验中，P-4501α仅在C-1位催化25-羟基-9，10-开环胆甾-5，7，10（19）-三烯-3 β-醇的羟基化，转换数为0.03 min−1。这个数字是相同的，从测量的催化活性在完整的线粒体，表明只有一个主要物种的cytochromeP-450发生在鸡肾线粒体。完整的线粒体中的细胞色素P-450浓度对雏鸡维生素D状态的完全反应提供了额外的证据，表明肾线粒体中存在的主要细胞色素P-450种类与维生素D代谢独特相关。
We describe the isolation of cytochromeP-4501αfrom chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount ofP-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which theP-4501αcould be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purifiedP-4501αtraveled as a single band in SDS gel electrophoresis with an apparentMr= 57 000. The absolute spectrum of theP-4501α(Fe3+) form gave a λmaxat 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure ofP-4501αwhich we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275–8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933–3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochromec(P-450) reductase complexes,P-4501αcatalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-ol at the C-1 position exclusively with a turnover number of 0.03 min−1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochromeP-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochromeP-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochromeP-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.