• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

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

Cytochrome b(5) (b(5)) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b(5) on CYP-catalyzed reactions, but also that of the apo-cytochrome b(5) (apo-b(5)). Therefore, the apo-b(5) protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b(5) was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5 alpha cells. The gene sequence was verified by DNA sequencing. The sequence coding b(5) was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b(5) was induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The b(5) protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE-Sepharose. Using such procedures, the homogenous preparation of apo-b(5) protein was obtained. Oxidized and reduced forms of the apo-b(5) reconstituted with heme exhibit the same absorbance spectra as native b(5). The prepared recombinant apo-b(5) reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b(5) is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b(5).

• MeSH
• apoenzymy genetika   metabolismus   MeSH
• chromatografie iontoměničová MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• cytochromy b5 genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• hem metabolismus   MeSH
• klonování DNA MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• naftoly metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH