Proton movement in reconstituted purple membrane of halobacteria: effects of pH and ionic composition of the medium.

Abstract Bacteriorhodopsin has been incorporated into sonicated phospholipid vesicles to form the functionally active proton pump. The kinetics of the light-induced proton uptake and of the subsequent release of protons in the dark have been analyzed according to a scheme which assumes the existence in bacteriorhodopsin of a light-dependent pathway responsible for an inhibition of the proton-pumping activity. The growth stage of proton movement obeys the empirical equation ln (1 − Δ Δ s ) = −k L t , where Δ and Δ s are the extent of proton uptake at time t of illumination and at the steady state, respectively. The release of protons in the dark stage follows the decay equation ln ( Δ Δ s ) = −k D t , where k D is a light-independent rate constant. With these definitions, the rate constant for the light-dependent proton-pumping inhibition is ( k L − k D ) = k I . The initial proton-pumping rate, R 0 , is obtained either directly from the “on rate” or from the steady-state equation R 0 = k L Δ s . We find that, in vesicles made from egg yolk phosphatidylcholine, changes in pH in the range 5–7 result in changes in both k I and R 0 , but have no significant effect on k D . The two empirical kinetic parameters, k I and R 0 , do not change to the same extent with pH, indicating a pH dependence of the coupling between them. When negatively charged phospholipids, e.g., phosphatidic acid, are incorporated into the phosphatidylcholine vesicles, the inhibition k I and the initial pumping rate, R 0 , are sensitive to pH changes. However, there are differences in the pH effects in the presence and absence of acidic phospholipids, and the coupling between pumping and its inhibition is no longer pH dependent. Thus, the presence in the bilayer of phospholipids with negatively charged head groups seems to stabilize the mechanism of coupling between R 0 and k I . Vesicles made from soybean phospholipids have been employed to study the effect of external ionic composition on the kinetics of the lightinduced proton movement. The K + of the external medium is replaced by other monovalent cations, at constant ionic strength and a fixed composition of the internal medium. Replacement of K + by Na + has no effect on the rate constants k D and k D but causes a gradual increase in the initial proton-pumping rate, R 0 . Replacement of K + by Cs + causes a marked decrease in the inhibition of the proton pumping measured by k I , without any concomitant effect on the other kinetic parameters. When most of the K + has been replaced by Cs + , pronounced effects are observed in all the parameters. Replacement of K + by Li + has no significant effect on any of the kinetic parameters, except when the K + Li + ratio is very low, in which case pronounced effects are again observed in all the kinetic parameters. These results and previous work from this laboratory, including the modification of the light-dependent proton-pumping inhibition by the charge of the bilayer, support a picture in which there is an indirect link between the primary proton-pumping activity and its associated inhibitory process in bacteriorhodopsin.