An investigation into the complex formation of membrane bound cytochrome b5 isolated from ovine liver microsomes

[摘要] ENGLISH ABSTRACT: Membrane bound cytochrome b5 is a ubiquitous protein with an average molecular weight of16 kDa. The protein is involved in a number of reactions providing electrons directly tocytochrome P450 enzymes or to other enzymes involved in lipid biosynthesis. It is alsoknown that the protein influences the activities of certain enzymes via an allosteric effect. Ithas been accepted in the literature that the cytochrome b5 exists primarily in the monomericform, however, recently it has been shown that it forms homomeric complexes in vivo. In thisstudy, we investigate the cytochrome b5 complex formation using a variety of analyticaltools. Cytochrome b5 was isolated from ovine liver microsomes and the purity verified usingsodium dodecyl sulphate polyacrylamide gel electrophoresis and electrospray ionisation massspectrometry. The latter analysis confirmed the presence of a single heme containing proteinwith Mr=15865 Da, while separation on the polyacrylamide gel revealed oligomeric complexformation with the tetrameric form the most prominent oligomer. Using different andparticularly harsh denaturing conditions we found that the observed oligomeric aggregatespersisted, indicating highly stable complexes. The most prominent tetrameric aggregate wasidentified to be cytochrome b5 by mass spectrometric sequencing. Further complex formationstudies, using a fluorescent dye (1-anilinonaphthalene-8-sulfonic acid) that interact withhydrophobic cavities formed during oligomerisation, provided evidence of protein assemblyin oligomeric complexes or aggregation. The formation of the cytochrome b5 complexes wasdependent on ionic strength and protein concentration. Previously it was shown that thehydrophobic membrane anchoring domain plays a pivotal role in the cytochrome b5'shomomeric complexes. Using a peptide (IITTIDSNSS), resembling a portion of this domain,together with circular dichroism we showed more organized structure present for the wildtypepeptide vs. a mutated control peptide (LLSSLKAVAV). A modified ELISA interactionassay also revealed that the wild-type peptide had a specific interaction with cytochrome b5,providing further evidence that the membrane anchoring domain plays a role in complexformation. These studies also indicated that a hydrogen bond network in this domain may beimportant for the formation of the homomeric complexes of cytochrome b5.

[发布机构] Stellenbosch University