H-DINITROPHENOL AND VALINOMYCIN(, K+, AND NA+ TRANSPORT ACROSS PHOSPHOLIPID VESICULAR MEMBRANE BY THE COMBINED ACTION OF PROTON UNCOUPLER 2,4)

The decay of the pH difference (Delta pH) across soyabean phospholipid vesicular membrane (created by temperature jump), by the combined act ion 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 f ollowing for the pH region of our interest (pH similar to 6 to pH simi lar 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. Th e rate constant associated with the transport of the ternary complex h as been estimated to be similar to 1.5 . 10(3) s(-1). (ii) The dissoci ation constant of the ternary complex Val-M(+)-DNP- in the membrane ar e 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 comp lexing 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 exp erimental strategy which enhances the Delta pH decay rate should also be a strategy for the efficient uncoupling of oxidative and photophosp horylation.