OBJECTIVES: Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of several cytochromes P450 (CYP). To elucidate the mechanism of such modulations it is necessary to evaluate not only the effect of native cyt b5, but also that of apo-cyt b5. To prepare apo-cyt b5, heme transfer from native cyt b5 to a protein with higher affinity toward the heme, the horse heart apo-myoglobin, was utilized. METHODS: Butanone extraction was employed to prepare apo-myoglobin. Apo-cyt b5 was separated from myoglobin by chromatography on DEAE-Sepharose. Mass spectrometry was utilized to characterize proteins eluted from DEAE- Sepharose. RESULTS: The prepared apo-myoglobin was incubated with the cyt b5 at pH 4.2 that is the optimal pH for heme transfer from cyt b5 into apo-myoglobin. The apo-cyt b5 protein was separated from myoglobin present in the reaction mixture by chromatography on a column of DEAE-Sepharose. Using such a procedure, 16% yield of apo-cyt b5 that did not contain any heme in its molecule was obtained from the native rabbit cyt b5. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native cyt b5. The prepared apo-cyt b5 reconstituted with heme can receive electrons from NADPH:CYP reductase. CONCLUSION: A biologically active apo-cyt b5 was prepared using transfer of heme from cyt b5 to horse heart apo-myoglobin by the procedure described here.
- MeSH
- absorpce MeSH
- apoproteiny chemie genetika MeSH
- butanony MeSH
- chromatografie MeSH
- cytochromy b5 chemie genetika metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- elektrony MeSH
- hem chemie MeSH
- hmotnostní spektrometrie MeSH
- koncentrace vodíkových iontů MeSH
- koně MeSH
- králíci MeSH
- molekulární sekvence - údaje MeSH
- myoglobin chemie genetika MeSH
- myokard chemie MeSH
- NADPH-cytochrom c-reduktasa chemie MeSH
- oxidace-redukce MeSH
- sekvence aminokyselin MeSH
- spektrální analýza MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- cytochrom P-450 CYP1A1 * MeSH
- cytochrom P-450 CYP1A2 * MeSH
- cytochromreduktasy MeSH
- elipticiny * farmakokinetika MeSH
- fytogenní protinádorové látky MeSH
- jaterní mikrozomy * MeSH
- klinické laboratorní techniky metody využití MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- NADPH-cytochrom c-reduktasa * MeSH
- protinádorové látky MeSH
- rozpřahující látky MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
