RESUMO
Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.
Assuntos
Sistema Enzimático do Citocromo P-450 , Microssomos Hepáticos/enzimologia , Análise Espectral Raman , Adsorção , Animais , Coloides , Citocromos , Eletrodos , Heme , Lipossomos/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Nonoxinol , Oxirredução , Fenobarbital/farmacologia , Polietilenoglicóis , Coelhos , Prata , SolubilidadeRESUMO
The binding of digitoxigenin to lecithin model membranes was investigated by application of electron spin- and nuclear magnetic resonance methods. A digitoxigenin spin-label derivative was found to bind specifically to the pseudohexagonal rigid lattice of lecithin membranes. The binding is accomplished by some of the structural features which are required for the biological activity of cardiac glycosides. Digitoxigenin has a procooperative effect on dipalmitoyl-lecithin membranes. The induction of lipid phase separation is discussed as a hypothetic molecular mechanism of action of cardiac glycosides on biological membranes.
Assuntos
Cardanolídeos/metabolismo , Digitoxigenina/metabolismo , Membranas Artificiais , Fosfatidilcolinas/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Sítios de Ligação , Digitoxigenina/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Cobaias , Modelos Estruturais , Contração Miocárdica/efeitos dos fármacos , Marcadores de Spin , TemperaturaRESUMO
The rotational diffusion of cytochrome P450SCC (CYP45011A1) was investigated in proteoliposomes measuring the time-resolved delayed fluorescence anisotropy of diiodofluorescein iodoacetamide covalently and specifically attached to Cys264 of P450SCC. Rotation strongly depends on the lipid composition of the membrane, especially on the cardiolipin content. In proteoliposomes having a lipid composition similar to the inner membrane of bovine adrenal mitochondria P450SCC is rotating uniaxially with a relaxation time phi rot about 53 microseconds and almost no immobile P450 is present. Addition of high KCl concentration has no effect. The results and the absence of any intramembrane particles observed by freeze-fracturing indicate that P450SCC probably exists in the liposomal membrane as oligomer not penetrating the bilayer. It is suggested to bind tightly within the outer monolayer with large parts exposed to the aqueous solution surrounding the membrane.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Proteolipídeos/metabolismo , Glândulas Suprarrenais/enzimologia , Animais , Bovinos , Fluoresceínas , Corantes Fluorescentes , Iodoacetamida/análogos & derivados , Cinética , Lipossomos , Fosfolipídeos , Espectrometria de FluorescênciaRESUMO
Cytochrome P450scc can be reconstituted successfully into large unilamellar phospholipid vesicles by a combined octylglucoside dialysis/adsorption method. Freeze-fracture electron microscopy was used to analyze the morphology, distribution, and protein topology of the cytochrome P450scc vesicles in dependence on lipid composition. Particles were observed only in close contact to the vesicle surface, probably representing tightly associated cytochrome P450scc at the outer vesicle surface. In cytochrome P450scc vesicles similar in lipid composition to the inner membrane of bovine mitochondria direct evidence by freeze-fracturing was found for a specific cytochrome P450scc-induced aggregation of the vesicles. The vesicle aggregation critically depends on the content of the specific mitochondrial membrane constituent cardiolipin. The aggregation and thus the intervesicular contacts were observed to be inhibited by both addition of anti-cytochrome P450scc IgG and adrenodoxin. Enzymatic reduction of cytochrome P450scc in the liposomal membrane by its electron transfer partners completely indicates an asymmetrical localization in/at the outer side of the bilayer membrane. It is suggested that vesiculation of the inner mitochondrial membrane may be a consequence of the characteristic cardiolipin-dependent cytochrome P450scc membrane topology: the cardiolipin binding, peripheral, non-bilayer-spanning integration as an oligomer in the outer leaflet of the membrane may play a role in the dynamics of formation and dissociation of intramitochondrial vesicles with a functional importance for steroidogenesis.
Assuntos
Cardiolipinas/química , Enzima de Clivagem da Cadeia Lateral do Colesterol/ultraestrutura , Lipossomos , Adrenodoxina/farmacologia , Animais , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Enzima de Clivagem da Cadeia Lateral do Colesterol/imunologia , Cromatografia em Gel , Técnica de Fratura por Congelamento , Glucosídeos , Imunoglobulina G/farmacologia , Lipossomos/química , Mitocôndrias Cardíacas/química , Fosfatidilcolinas , FosfatidiletanolaminasRESUMO
Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.