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

The decay of the pH difference (Delta pH) 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 similar to 6 to pH similar to 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 similar to 1.5 . 10(3) s(-1). (ii) The dissociation constant of the ternary complex Val-M(+)-DNP- in the membrane are similar to 1 mM for M(+) = K+ and similar to 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 Delta pH decay rate should also be a strategy for the efficient uncoupling of oxidative and photophosphorylation.