Association of bacteriorhodopsin-containing phospholipid vesicles with phospholipid-impregnated Millipore filters

Abstract

A photopotential is generated upon illumination of a phospholipid-impregnated Millipore filter to which are bound phospholipid vesicles, containing bacteriorhodopsin isolated from Halobacterium halobium. In this study, experiments are described that show how the bacteriorhodopsin-dependent photo effect is influenced by factors that have an affect on the association of bacteriorhodopsin-containing vesicles with the filter. The following conclusions are drawn: 1. 1. The photopotential generated during a short flash of light is directly proportional to the number of bacteriorhodopsin vesicles associated with the filter. 2. 2. The number of bacteriorhodopsin molecules that contribute to the photo effect increases with time according to a hyperbolic relationship, reaching a maximum after prolonged incubation. 3. 3. The increase of the photo effect with time can be ascribed to a time-dependent association of bacteriorhodopsin vesicles with the filter. The maximum value of the photo effect is due to a saturation of the filter with bacteriorhodopsin vesicles. 4. 4. The rate of association of bacteriorhodopsin vesicles with the filter is directly proportional to the vesicle concentration in the aqueous medium. The maximal number of vesicles that becomes associated with the filter after prolonged incubation is independent of this vesicle concentration. 5. 5. Both the rate of association of bacteriorhodopsin vesicles with the filter and the maximal photo effect calculated by extrapolation to infinite incubation time increase with the Ca 2+ concentration in the medium, and reach a plateau at approximately 40 mM Ca 2+. This suggests that Ca 2+ has an effect on the number of bacteriorhodopsin vesicles that can associate with the filter. 6. 6. The maximal photo effect is decreased by the simultaneous presence in the aqueous medium of untreated bacteriorhodopsin vesicles and of vesicles in which bacteriorhodopsin has been inactivated by treatment at elevated pH or with KBH 4. From this we conclude that inactivated bacteriorhodopsin vesicles can still associate with the filter in a way comparable to untreated bacteriorhodopsin vesicles. On the other hand, pure phospholipid vesicles only affect the rate of association of bacteriorhodopsin vesicles with the filter but have no effect on the maximal photo effect calculated by extrapolation to infinite time.