Bacteriorhodopsin reconstituted vesicles

Preparation and dialysis of the vesicles in buffers that contained 20-mM K2HP04 and NaH2P04 instead of 2 mM, which produced smaller concentration ratios of K+ across the membrane yielded results that were similar, but the effects were smaller. Figure 4 shows the effect of the electrical potential on the CD intensity of bacteriorhodopsin reconstituted vesicles. The potentials on the abscissa are calculated assuming unity for K+ transport number the sign refers to the polarity of the interior of the vesicles. 

FIGURE 21.28 The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis. 

Further evidence for the light-driven proton translocation by bacteriorhodopsin was obtained by Racker and Stoeckenius who carried out reconstitution ofthe purple membrane into phospholipid vesicles. In the light, the reconstituted vesicles took up protons from the exterior at a rate of 50-200 ng per mg of bacteriorhodopsin, and released them in the dark. The rate of proton uptake in the fight and release in the dark was accelerated by the addition ofvalinomycin, while uncouplers of oxidative phosphorylation abolished the uptake of protons altogether. Note that the direction of proton transport from outside to the inside of the vesicle reported by the authors was opposite to that observed in intact cells, the possibility that bacteriorhodopsin might be oppositely oriented in cells and vesicles, was subsequently confirmed by freeze-etch electron microscopy. 

The purple membrane fragments that contained dark-adapted bacteriorhodopsin were used to form reconstituted vesicles with a mixture of phospholipids that contained 80% egg phosphatidyl choline and 20% bovine phosphatidylserine (Lipid Products, Nuttfield, England). The lipids in chloroform and methanol solutions were mixed to the desired composition, dried in a stream of nitrogen, placed in a 0.1-torr... 

Figure 4. The dependence of the ellipticity of bacteriorhodopsin reconstituted in vesicles on the electric diffusion potentials. The ordinate is the decrease of the CD signal at 210 nm induced by 10 7-M valinomycin. The abscissa is the electrical potentials calculated by the Nemst equation. The sign indicates the polarity inside the vesicle.

Form reconstituted vesicles containing lactose permease and bacteriorhodopsin, with each oriented in the membrane so that its cytoplasmic face is toward the inside of the vesicle. Illumination of such vesicles will cause hydrogen ions to be extruded by bacteriorhodopsin. The resulting gradient of hydrogen ions will then drive the entry of lactose by its permease. 

Uruga, T., Hamanaka, T., Kito, Y, Uchida, 1., Nishimura, S., and Mashimo, T, Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles, Biophys. Chem., 41,2, 157-168, 1991. 

Krupinski, J., G. G. Hammes, Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles. Biochemistry, 1985,24,6963-6972. Zgoda, M., S. Petri, Surfactants from copolymers of propylene and ethylene oxides. I Dispersion of weight-average molecular weights (in Poli.sh), Chem. Anal. (Warsaw), 1986, //, 577-5%. 

Figure 18.26. Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated. The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion.

A model system for oxidative phosphorylation can be constructed with proton pumping in the absence of electron transport. The model system consists of reconstituted membrane vesicles, mitochondrial ATP synthase, and a proton pump. The pump is bacteriorhodopsin, a protein found in the membrane of halobacteria. The proton pumping takes place when the protein is illuminated (Figure 20.17). 

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