Thesis (MSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Membrane bound cytochrome b5 is a ubiquitous protein with an average molecular weight of
16 kDa. The protein is involved in a number of reactions providing electrons directly to
cytochrome P450 enzymes or to other enzymes involved in lipid biosynthesis. It is also
known that the protein influences the activities of certain enzymes via an allosteric effect. It
has been accepted in the literature that the cytochrome b5 exists primarily in the monomeric
form, however, recently it has been shown that it forms homomeric complexes in vivo. In this
study, we investigate the cytochrome b5 complex formation using a variety of analytical
tools. Cytochrome b5 was isolated from ovine liver microsomes and the purity verified using
sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrospray ionisation mass
spectrometry. The latter analysis confirmed the presence of a single heme containing protein
with Mr=15865 Da, while separation on the polyacrylamide gel revealed oligomeric complex
formation with the tetrameric form the most prominent oligomer. Using different and
particularly harsh denaturing conditions we found that the observed oligomeric aggregates
persisted, indicating highly stable complexes. The most prominent tetrameric aggregate was
identified to be cytochrome b5 by mass spectrometric sequencing. Further complex formation
studies, using a fluorescent dye (1-anilinonaphthalene-8-sulfonic acid) that interact with
hydrophobic cavities formed during oligomerisation, provided evidence of protein assembly
in oligomeric complexes or aggregation. The formation of the cytochrome b5 complexes was
dependent on ionic strength and protein concentration. Previously it was shown that the
hydrophobic membrane anchoring domain plays a pivotal role in the cytochrome b5’s
homomeric complexes. Using a peptide (IITTIDSNSS), resembling a portion of this domain,
together with circular dichroism we showed more organized structure present for the wildtype
peptide vs. a mutated control peptide (LLSSLKAVAV). A modified ELISA interaction
assay 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 complex
formation. These studies also indicated that a hydrogen bond network in this domain may be
important for the formation of the homomeric complexes of cytochrome b5. / AFRIKAANSE OPSOMMING: Membraan-gebonde sitochroom b5 is ’n alomteenwoordige proteïen met ’n gemiddelde
molekulêre massa van 16 kDa. Die proteïen is betrokke in reaksies waar dit elektrone direk
aan sitochroom P450 ensieme verskaf, sowel as ensieme betrokke in lipiedbiosintese. Dit is
ook bekend dat die proteïen die aktiwiteite van sekere ensieme via ’n allosteriese effek
beïnvloed. Dit is geredelik in die literatuur aanvaar dat sitochroom b5 as ’n monomeer
voorkom, maar daar is kort gelede gerapporteer dat homomeriese komplekse in vivo vorm. In
hierdie studie is die sitochroom b5-kompleksvorming ondersoek deur gebruik te maak van
verskeie analietiese metodes. Sitochroom b5 is vanuit skaaplewer mikrosome geïsoleer en die
suiwerheid met behulp van natrium-dodesiel-sulfaat-poliakrielamied-gel-elektroforese en
elektrosproei-ionisasie massa-spektrometrie geverifieer. Met die laasgenoemde bevestig dat
’n enkele heem-bevattende proteïen met Mr =15865 teenwoordig was, terwyl poliakrielamied
gel-skeiding kompleksvorming getoon het, met tetrameer as die mees prominente oligomeer.
Deur verskeie denaturerings kondisies, intsluitend besondere kondisies, is gevind dat hierdie
aggregate behoue bly, wat baie stabiele oligomere aandui. Die mees prominente tetrameriese
aggregaat is as sitochroom b5 geïdentifiseer met behulp van massa spektrometriese
volgordebepaling. Kompleksvorming is verder bewys deur ’n verdere ondersoek met behulp
van ’n fluoresserende kleurstof (1-anilinonaftaleen-8-sulfoonsuur) wat met die hirdofobiese
holtes, wat vorm tydens oligomermerisasie, interaksie het. Die kompleksvorming was
afhanklik van ioniese sterkte, sowel as proteïenkonsentrasie. Voorheen was dit bewys dat die
deurslaggewende faktor in die vorming sitochroom b5 se homomeriese komplekse die
hidrofobiese membraan-anker-domein is. Deur gebruik te maak van ’n peptied
(IITTIDSNSS) wat lyk soos ’n gedeelte van hierdie domein, tesame met sirkulêre
dichroisme, is gewys dat meer georganiseerde struktuur teenwoordig was vir die wilde tipe
peptied vs. ’n gemuteerde kontrole peptied (LLSSLKAVAV). ’n Gemodifiseerde ELISAinteraksie-
essai het ook getoon dat die wilde-tipe peptied spesifieke interaksie met
sitochroom b5 het, ’n verdere bewys dat hierdie membraan-anker-domein ’n rol speel in
kompleksvorming. Hierdie studies het ook aangedui dat ’n waterstofbinding netwerk in die
domein belangrik kan wees vir die vorming van die homomeriese komplekse van sitochroom
b5.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85653 |
| Date | 12 1900 |
| Creators | Adriaanse, Craig Vernon |
| Contributors | Swart, P., Rautenbach, M., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 88 p. : ill. |
| Rights | Stellenbosch University |
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