H+, K+, and Na+ transport across phospholipid vesicular membrane by the combined action of proton uncoupler 2,4-dinitrophenol and valinomycin

AbstractThe decay of the pH difference (ΔpH) across soyabean phospholipid vesicular membrane (created by temperature jump), by the combined action of valinomycin and 2,4-dinitrophenol (DNP) has been monitored with the help of fluorescence from pyranine entrapped inside the vesicles under a variety of concentration conditions. The results suggest the following for the pH region of our interest (pH ∼ 6 to pH ∼ 8): (i) The rate limiting step in the proton transport cycle is not the transport of proton as DNPH, but the back transpsort of DNP− and the alkali metal ion M+ as Val-M+-DNP− across the membrane. The rate constant associated with the transport of the ternary complex has been estimated to be ∼ 1.5 · 103 s−1. (ii) The dissociation constant of the ternary complex Val-M+-DNP− in the membrane are ∼ 1 mM for M+K+ and ∼ 0.001 mM for M+Na+. (iii) The reduction in the cation selectivity of valinomycin on complexing with DNP− is much more than that observed with the anionic form of carbonyl cyanide m-chlorophenylhydrazone (CCCP). The results also provide a verification of a corollary of Mitchell's hypothesis: an experimental strategy which enhances the ΔpH decay rate should also be a strategy for the efficient uncoupling of oxidative and photophosphorylation