Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver
Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver
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DOI：
10.1016/s1357-2725(98)00099-5
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发表时间：
1999-02-01
影响因子：
4
通讯作者：
Çakir, D
中科院分区：
文献类型：
--
作者：
Arinç, E;Çakir, D
Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons fromNADH-cytochrome b5 reductase to cytochrome c was investigated. The K-m and V-max values were calculated to be 0.088 mu M cytochrome b5 and 315.8 mu M cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K-m and V-max values were determined to be 5 mu M cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K-m and V-max values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE,When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K-m and V-max values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mu mol ferricyanide reduced/min/mg enzyme, respectively. The K-m and V-max values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mu mol ferricyanide reduced/min/mg enzyme, respectively. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.
