Location of two sulfhydryl groups in the rhodopsin molecule by use of the spin label technique

Sulfhydryl groups of membrane-bound rhodopsin are studied with the spin label technique by using five maleimide derivative probes of different lengths. Two sulfhydryl groups are titrated per molecule of rhodopsin. These groups are located in protected but probably different environments, less then 12 Å away from the aqueous phase. A distance of about 37 Å is measured between the two groups. These results are consistent with a model in which the two groups would be located close by the external surface of the protein but embedded within the membrane layer, the strong immobilization of the label molecules resulting from phospholipid-protein interactions.     

Fluid lipid fraction in rod outer segment membrane.

Rod outer segment membrane is analyzed using the spin resonance label technique by means of two probes. The solubility of the first label,2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Quantum efficiency of light-driven proton extrusion in Halobacterium halobium. pH dependence.

The quantum yield for light-induced proton extrusion in Halobacterium halobium cells pretreated with an ATPase inhibitor was measured between pH 5 and 9 using two separate spectrophotometric techniques. The transmittance of the cell suspension was measured with a spectrometer equipped with "end-on" photomultipliers, whereas the reflectance was measured using a light-integrating sphere. The potentialities of the two techniques are critically compared. These measurements are used to evaluate the intensities of light absorbed by the cells. Since the initial rates of proton release into the extracellular medium were simultaneously measured, the quantum yield values [QY(H+)] could be determined. The results obtained with the two techniques are in reasonable agreement. QY(H+) is 0.64 at pH 5.9 and decreases gradually to 0.28 at alkaline pH values.