Outer mitochondrial membrane cytochrome b 5 is an isoform of microsomal membrane cytochrome b 5 . In rat testes the outer mitochondrial membrane cytochrome b 5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b 5 is undetectable. Outer mitochondrial membrane cytochrome b 5 present in the testis was localized in Leydig cells with cytochrome P-450 17␣ , which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b 5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b 5 stimulated the cytochrome P-450 17␣ -catalyzed reactions, 17␣-hydroxylation and C17-C20 bond 1 in the endoplasmic reticulum (ER), and the other is outer mitochondrial membrane cytochrome b 5 (OMb) (1-3). They consist of the following three domains: (a) the amino-terminal hydrophilic, (b) medial hydrophobic, and (c) carboxyl-terminal hydrophilic domains. A protoheme is bound to the first domain, which is highly conserved between b 5 and OMb with 70% identity (4, 5). The hydrophobic domain, consisting of about 20 amino acid residues, is embedded in the lipid bilayer and functions for the insertion of proteins into the membranes as tail-anchored proteins (6). The carboxyl-terminal 10 amino acid residues of b 5 are exposed to the luminal side of the ER cisterna (7,8) and are required for the targeting of the cytochrome to the organelle (9). The visible spectroscopic properties of the reduced forms are characteristic of the b 5 -type hemoproteins. OMb and b 5 are spectrographically indistinguishable from each other due to the highly conserved heme-binding portion (1, 2). OMb and b 5 have a similar mobility on SDS-PAGE and are co-purified unless specific precaution was taken in the purification procedures. Antibodies directed against b 5 that had been purified without the removal step of OMb cross-react with OMb. In some case, cross-reactions are observed even if a highly purified form of OMb or b 5 was immunized in rabbits. These facts made the discrimination between OMb and b 5 difficult. The most convincing way of the discrimination is use of the specific antibodies. We have such antibodies in a large collection of anti-OMb and anti-b 5 antibodies.Because b 5 is a hemoprotein with sixth co-ordination, it is incapable of activating an oxygen molecule as cytochrome P-450 does. One of its physiological functions is an electron transfer to terminal oxidases such as stearyl-CoA desaturase (10). Some evidence also suggests that b 5 functions as a modifier for some cytochrome P-450-catalyzed reactions although its mechanism is not clear (11,12). For example, b 5 stimulates the 6␤-hydroxylation of testosterone and nifedipine oxidation by recombinant CYP3A4 (13). It also augments C17-C20 lyase activity of pig, guinea pig, and human P-450 17␣ leading to predominant formation of androgens (androstenedione and de...

Cytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER-membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.