Anions and cations have long been recognized to be capable of modifying the functioning of various membrane-related physiological processes. Here, a fluorescent ratio method using the styrylpyridinium dyes, RH421 and di-8-ANEPPS, was applied to determine the effect of a range of anions and cations on the intramembrane dipole potential of dimyristoylphosphatidylcholine vesicles. It was found that certain anions cause a decrease in the dipole potential. This could be explained by binding within the membrane, in support of a hypothesis originally put forward by A. L. Hodgkin and P. Horowicz [1960, J. Physiol. (Lond.) 153:404-412.] The effectiveness of the anions in reducing the dipole potential was found to be ClO4- > SCN- > I- > NO3- > Br- > Cl- > F- > SO42-. This order could be modeled by a partitioning of ions between the membrane and the aqueous phase, which is controlled predominantly by the Gibbs free energy of hydration. Cations were also found to be capable of reducing the dipole potential, although much less efficiently than can anions. The effects of the cations was found to be trivalent > divalent > monovalent. The cation effects were attributed to binding to a specific polar site on the surface of the membrane. The results presented provide a molecular basis for the interpretation of the Hofmeister effect of lyotropic anions on ion transport proteins.

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

The kinetics of the E 2 f E 1 conformational change of unphosphorylated Na + ,K + -ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E 2 conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/τ, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 ( 3 s -1 in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na + to the enzyme in the E 2 state with a dissociation constant of 31 ( 7 mM, which induces a subsequent rate-limiting conformational change to the E 1 state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/τ for both the NaCl and choline chloride titrations. The results suggest that Na + ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E 2 f E 1 conformational transition by interaction with the E 2 state.The Na + ,K + -ATPase is known to play a fundamental role in numerous physiological processes, e.g., nerve, kidney, and heart function. Its activity in the cell must, therefore, be under tight metabolic control. A major site of regulation of the enzyme at the molecular level must be at its rate-determining steps, since only changes in the rates of these steps will result in any significant change in the overall turnover number of the enzyme.The kinetics of the Na + ,K + -ATPase are generally described in terms of the Albers-Post model (1, 2), a simplified version of which is shown in Figure 1. This simple model considers two conformations of the enzyme, E 1 and E 2 , which can be either in a phosphorylated or an unphosphorylated state. The model, furthermore, describes a consecutive mechanism of Na + and K + ion transport across the membrane, whereby Na + ions normally bind from the cytoplasm and K + ions from the extracellular fluid. The location of the rate-determining steps within this cycle and the determination of rate constants for the various steps have been the subject of an intense research effort by many groups over the past thirty years. Although several different reaction steps have been discussed as possible candidates for the rate-determining step of the enzyme, there now appears to be conclusive evidence and an overall consensus that under physiological conditions it is in fact the E 2 f E 1 transition of unphosphorylated enzyme (3).It is known that the enzyme can be ...