(A) In concert with kinetic studies using intact cells of Halobacteria (Z01 HL 00401-32 LCB), we are studying proton-pumping capabilities of these cells. We find that the cells do not pump protons to the external medium upon illumination as expected. Instead, there is an small initial proton-uptake followed by a small amount of proton-release. In these intact cells, proton-release by the bacteriorhodopsin (BR) pump is counterbalanced by proton uptake due to ATP-synthesis and by H+/K+ antiport, driven by a large existent K+ gradient. Further complications are due to proton extrusion in the dark, driven by respiration and ATP hydrolysis. Proton-pumping by these cells can be demonstrated after an overnight pre-incubation with HCN to inhibit respiration, DCCD to inhibit ATP-synthesis and breakdown, and a small amount of nigericin to dissipate the K+ gradient and expedite charge neutralization by stimulation of the H+/K+ antiport. Light-activated proton pumping in fresh cells requires treatment with all three of these inhibitors and ionophores. In aged cells, where the K+ gradient and respiratory activity has been greatly diminished, net light-driven proton-pumping can be established by pre-incubation with DCCD alone. (B) We have tried to identify the precise photointermediate transitions that are responsible for voltage formation across the cell membrane and to determine the relative electrogenicity in the decays of the M-fast and M-slow photocycle intermediates. To accomplish this we are using the real-time kinetic, voltage-measurement techniques developed by Lel Drachev. We have been hampered by the fact that the kinetics of voltage formation are much slower than the kinetics of photocycle transitions established by optical measurements using our rapid scan multichannel techniques. One important difference in the two procedures is the required use of decane and non-native phospholipids in the voltage-measuring system to attach the purple membrane patches to a Teflon membrane support. Much time was spent in investigating the possible roles of the lipids and/or decane in slowing the photocycle kinetics, with no success. Because of our findings that voltage on the membrane can seriously slow photocycle kinetics and that fast kinetics can be reestablished with the energy uncoupler, CCCP (Z01 HL 00401-32 LCB), we tried to speed the kinetics in the Drachev system by addition of varying amounts of CCCP. We find that, indeed, kinetics of voltage-formation are speeded by addition of CCCP. One problem is that normal kinetics will require an amount of CCCP sufficient to prevent any voltage formation. Experiments are now proceeding to titrate both kinetics and magnitudes of voltage formation with the aim of establishing a direct relationship between kinetic steps in the electrogenic processes and those of photocycle transitions in the optically monitored system. In addition to accomplishing our aims, these findings will be important to other laboratories who are using the Drachev system to correlate steps in different kinetic processes with electrogenicity.
|Hendler, R W; Drachev, L A; Bose, S et al. (2000) On the kinetics of voltage formation in purple membranes of Halobacterium salinarium. Eur J Biochem 267:5879-90|
|Joshi, M K; Bose, S; Hendler, R W (1999) Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium. Biochemistry 38:8786-93|