RESUMEN
Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4 M urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.
Asunto(s)
Fabaceae/química , Canales Iónicos/aislamiento & purificación , Mitocondrias/química , Plantas Medicinales , Porinas/aislamiento & purificación , Secuencia de Aminoácidos , Fabaceae/metabolismo , Fabaceae/ultraestructura , Canales Iónicos/química , Membrana Dobles de Lípidos/química , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfatidilcolinas/química , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Porinas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Semillas/química , Semillas/metabolismo , Semillas/ultraestructura , Alineación de Secuencia , Análisis de Secuencia de Proteína , Canales Aniónicos Dependientes del VoltajeRESUMEN
Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel beta-sheet. The orientation of the beta-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I beta-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.