Rhodopsin--phospholipid complexes in apolar environments: photochemical characterization.
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Biomedical subjects
Publications and source records attributed to M Montal.
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A quasi-short-circuit (tunable voltage clamp) measurement method with microsecond time resolution was applied to a bacteriorhodopsin model membrane formed by a novel interfacial technique. A new component (B1) of the displacement photocurrent was recorded: it has no detectable latency at an instrumental time constant of 1.5 museconds, and persists at 5 degrees C. In addition, a slower component (B2) of opposite polarity inhibited by low temperature (5 degrees C) and low pH (pH = 3.0) was recorded. The technique is very sensitive for the study of fast capacitative photoresponses in model membranes, and allows the detection of charge displacements in bacteriorhodopsin associated with distinct stages of the photochemical transformation.
Planar lipid bilayers containing reaction centers from Rhodopseudomonas sphaeroides R-26 were formed by apposing two reaction center-lipid monolayers formed from a reaction center-lipid complex in hexane. Secondary donors (cytochrome c) and acceptors (ubiquinone-0) were added on opposite sides of the membrane. Upon illumination, this system generated transient and steady-state voltages and currents. The wavelength dependence of the photoresponse matched the absorption spectrum of reaction centers. A simple model based on the transfer of charges across the membrane that explains the salient features of the photoresponse is presented.
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 approximately 80 A 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 micron and approximately 40% into vesicles larger than 1 micron.
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Rhodopsin has been incorporated into planar lipid bilayer membranes. The effect of light is to increase the bilayer permeability in a pattern consistent with the formation of a transmembrane channel of about 10 A diameter. A model of visual excitation based on a light-activated and voltage-sensitive channel is presented.
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The interaction of Ca++ with acidic phospholipids in black lipid films and lipid bilayers formed from two monolayers was studied by measuring their physical stability and conductance. It was found that the addition of CaCl2 to only one side of lipid bilayers formed from phosphatidylserine or cardiolipin does not appreciably change these parameters. In contrast, black films are unstable to the asymmetric addition of CaCl2. Therefore, the destabilizing effect of Ca++ cannot be attributed to a surface charge difference. The only variation in composition between both bilayer membranes, namely the solvent content of the bilayer, seems to be responsible for the distinctive effect of Ca++. A tentative explanation is presented.
A model membrane was developed in which interfacial layers of rhodopsin were reoriented onto one side of a thin Teflon film separating two aqueous compartments. Flashes evoked fast photoelectric signals (1 ms) that originated from capacitative charge displacements of oriented rhodopsin upon bleaching. The photoelectric responses of rhodopsin in the model membrane are compared with the early receptor potential of photoreceptor cells; it is concluded that the signals in both systems originate from the same mechanism.
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Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.
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Planar bilayer membranes were formed from monolayers of a series of mono-unsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 muF/cm-2 which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride. The specific capacity (Cm) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the Cm increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with n-decane as a solvent.
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