Purification and Properties of a Cytochrome b5-Like Hemeprotein from Mitochondrial Outer Membranes of Rat Liver
- 1 November 1977
- journal article
- research article
- Published by Oxford University Press (OUP) in The Journal of Biochemistry
- Vol. 82 (5), 1257-1266
- https://doi.org/10.1093/oxfordjournals.jbchem.a131813
Abstract
Upon solubilization with sodium cholate, the cytochrome b5-like hemeprotein associated with the outer membranes of rat liver mitochondria (abbreviated here as OM-cytochrome b5) could be preferentially released from sonicated mitochondrial outer membrane preparations. The OM-cytochrome b5 thus solubilized was highly purified by DEAE-cellulose, DEAE-Sepharose CL-6B, hydroxylapatite column chromatography in the presence of 0.5% sodium cholate and the isoelectric focusing method. Sepharose 6B gel chromatography in the presence of 6 M guanidine hydrochloride and Sephadex G-75 gel chromatography in the presence of 0.5% sodium dodecyl sulfate (SDS) indicated a monomeric molecular weight of about 16,000 for the detergent-solubilized OM-cytochrome b5. However, in aqueous solutions, this cytochrome occurs as a stable polymeric species since its molecular weight has been found to be approximately 60,000 even in 0.5% sodium cholate. The absorption spectrum of reduced OM-cytochrome b5 in the visible region closely resembled that of microsomal cytochrome b5, however, the OM-cytochrome b5 showed a broad, symmetrical α-band with a maximum around 557 nm. A high level of NADH-cytochrome c reductive activity was detected by the addition of both the purified L-kynurenine 3-hydroxylase [EC 1.14.13.9] and OM-cytochrome b5 preparation to a sonicated phospholipids liposome bilayer, although only the purified monooxygenase or OM-cytochrome b5 could not catalyze the reduction of cytochrome c by NADH. The rate of NADH-cytochrome c reduction catalyzed by the monooxygenase and this hemeprotein in the presence of PC liposomes was affected by high concentrations of rotenone or dicumarol.Keywords
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