The studies on the decay of the pH difference, delta pH, across soyabean phospholipid vesicular membrane have shown that the rates of net proton transport and the associated Li+ and Na+ ion transport across the membrane can be enhanced by the combined action of gramicidin, valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) in K(+)-free vesicle solutions. The data obtained under different experimental conditions suggest that this enhancement is a consequence of facilitation of CCCP- transport (1) by complexing CCCP- with the highly membrane permeant valinomycin without the metal ion bound to it and (2) by the associated Li+ or Na+ transport through the gramicidin channel such that no net charge is transported across the membrane. The dissociation constant of the weak valinomycin-CCCP- complex has been estimated to be > 200 mM in the membrane. The delta pH in these experiments were created by temperature jump.
The p-benzosemiquinone radical anion has served as a model system in numerous electron spin resonance liquid-phase relaxation studies. An inconsistency exists of more than an order of magnitude between measured spin-lattice relaxation times T1e and calculated values based on the spin-rotational mechanism if the rotational correlation time τθ that is required in the calculation is obtained from linewidth measurements. This paper presents a number of experiments designed to contribute to the solution of this problem. The rigid-limit g values of tetrahalogenated benzosemiquinone anions have recently become available, permitting the study of the effect of Zeeman anisotropy in a relatively homologous series of compounds. 13C labeled tetrachlorobenzosemiquinone anion (TSCQ−) has additionally been prepared in order to determine the dependence of linewidth on the 13C nuclear quantum number. Linewidth and continuous wave (cw) saturation measurements have been made from 1.0 to 35 GHz. Saturation-recovery pulse measurements of T1e have been made at 9 GHz. Solvent viscosity and temperature have been varied. It is established that an expression of the form [T−11e=AT/η+B exp(−ΔE/RT)] is required to fit the data in hydrogen-bonding solvents with both terms arising from spin-rotation. Values of τθ were determined in five different ways for TCSQ−. The variation is fivefold in ethanol and tenfold in n-butanol. The discrepancy that motivated the research has not been fully resolved, but the data suggests the existence of an additional line broadening mechanism arising from the superposition of signals from radicals in a distribution of relatively long-lived local environments.
Enhancement of delta pH relaxation rate by the combined action of valinomycin (VAL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been studied under a variety of concentration conditions in soyabean phospholipid (SBPL) vesicles after creating a pH gradient across the vesicular membrane delta pH by temperature jump. After taking note of the changes by VAL and CCCP induced membrane disorder (using nigericin and monensin mediated delta pH decay as probes) the following could be inferred about the mechanism of enhancement of delta pH decay rate: (i) in solutions containing KCl, the rate limiting species have been identified to be (a) Val-K(+)-CCCP-, at low [Val]0 and [CCCP]0 (with translocation rate constant k2 approximatley 3.2 x 10(3) s-1); (b) CCCPH, at high [Val]0 (with translocation rate constant k1 approximately 2 x 10(5) s-1); (c) the neutral valinomycin species Val, at high [CCCP]0. (ii) In solutions containing NaCl, in our concentration range, the rate limiting species are Val-Na(+)-CCCP-. (iii) The apparent dissociation constant K*M of Val-M+ decreases with pH in SBPL vesicles but is independent of pH in vesicles prepared from PC + 6% PA. (iv) The differences in the ionic strength dependencies of kinetic data shows that the environments of Na+ and K+ binding sites on VAL are different. (v) In vesicle solutions containing 100 mM MCl, the cation selectivity of VAL (towards K+ in preference to Na+) is reduced when CCCP- is already bound to it in the membrane. The CCCP- dissociation constant of Val-M(+)-CCCP- is smaller with M+ = Na+ (approximatley 0.22 mM at 100 mM NaCl) when compared to that with M+ = K+ (approximately 2 mM at 100 mM KCl). Attributing these differences to the differences in electrostatic interaction between CCCP- and M+ in Val-M(+)-CCCP-, we can say that CCCP- binds closer to the Na+ binding site than to the K+ binding site on VAL.
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