SHEEP ADRENAL CYTOCHROME b₅: ACTIVE AS A MONOMER OR A TETRAMER IN VIVO?

Cytochrome b(5) (cyt b(5)) is an ubiquitous hemoprotein also associated with microsomal cytochromes P450. It has been reported that cyt b(5) influences cytochrome P450-dependent catalyses through electron transport as well as direct protein-protein interactions. To investigate the influence of cyt b(5) on ovine adrenal steroidogenesis, we isolated and characterized cyt b(5) from ovine liver. The molecular mass of the purified protein was 15,260 as determined by electrospray mass spectrometry. SDS-Polyacrylamide gel electrophoresis, even after stringent detergent and mercaptoethanol pretreatment, indicated multimeric forms of the protein, the most prominent being the tetramer (+/-60kDa) with minor bands corresponding to the monomer (+/-16kDa) and dimer (+/-30kDa). Trypsin treatment of cyt b(5) resulted in a truncated enzyme with a molecular mass of +/-10kDa. The aggregation of cytochrome b(5) was abolished by the tryptic removal of the membrane binding region. In Western blot analyses antibodies against the truncated protein recognised only this low molecular mass form and not the full length cyt b(5), or any of the higher molecular complexes, showing the involvement of the membrane binding domain of the protein, not only in aggregation, but also in the quaternary structure which determines epitope presentation for antibody production. Immunoblot analyses of sheep adrenal microsomes with the anti-truncated cyt b(5) antibody were also negative. Immunoblot analyses and immunocytochemistry of adrenal tissue with antibodies against the full length cyt b(5) indicated that the tetrameric form of the protein was in all probability the dominant specie in vivo.