Application to Rhodopsin
نویسندگان
چکیده
A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles 8 0 .~ in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0 .5 /xm and 4 0 % into vesicles larger than 1 /xm.
منابع مشابه
Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin
A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aq...
متن کاملLight-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.
A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Mod...
متن کاملInhibitory Peptide of Mitochondrial μ-Calpain Protects against Photoreceptor Degeneration in Rhodopsin Transgenic S334ter and P23H Rats
Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated...
متن کاملSynchronizing multiphasic circadian rhythms of rhodopsin promoter expression in rod photoreceptor cells.
Endogenous circadian clocks regulate day-night rhythms of animal behavior and physiology. In zebrafish, the circadian clocks are located in the pineal gland and the retina. In the retina, each photoreceptor is considered a circadian oscillator. A critical question is whether the individual circadian oscillators are synchronized. If so, the mechanism that underlies the synchronization needs to b...
متن کاملThe contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin
Most of the photoreceptors of the fly compound eye have high sensitivity in the ultraviolet (UV) as well as in the visible spectral range. This UV sensitivity arises from a photostable pigment that acts as a sensitizer for rhodopsin. Because the sensitizing pigment cannot be bleached, the classical determination of the photosensitivity spectrum from measurements of the difference spectrum of th...
متن کامل