RESUMO
The time-resolved kinetics of the KdpFABC complex solubilized in Aminoxide WS-35 was investigated by ATP concentration jump experiments. ATP was photoreleased from its inactive precursor, caged ATP, and charge movements in the membrane domain of the KdpFABC were detected by the electrochromic dye RH421. At low ATP concentrations, the ATP binding step became rate-limiting with an apparent, pH-independent ATP binding affinity of ~70 µM. At saturating ATP concentrations, the rate-limiting step is the conformational transition (E1-P â P-E2) with a rate constant of ~1.7 s(-1) at 20 °C that was independent of K(+) concentration. This observation together with the detected fluorescence decrease indicates that K(+) (or another positive ion) is bound in the membrane domain after enzyme phosphorylation and the conformational transition to the P-E2 state. pH dependence experiments revealed different roles of H(+) in the transport mechanism. Two different functions of protons for the ion pump must be distinguished. On one hand, there are electrogenically bound "functional" protons, which are not transported but prerequisite for the performance of the ATP-driven half-cycle. On the other hand, protons bind to the transport sites, acting as weak congeners of K(+). There possibly are noncompetitively bound protons, affecting the enzyme activity and/or coupling between KdpA and KdpB subunits. Finally, the recently proposed Post-Albers model for the KdpFABC complex was supplemented with stoichiometry factors of 2 for K(+) and 3 for H(+), and additional inhibitory side reactions controlled by H(+) were introduced, which are relevant at pH <6.5 and/or in the absence of K(+).
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Transporte de Cátions/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Modelos Químicos , Complexos Multienzimáticos/química , Prótons , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons/fisiologia , Cinética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Potássio/química , Potássio/metabolismoRESUMO
An open membrane preparation containing SR Ca-ATPase was prepared from sarcoplasmic-reticulum vesicles to study the ion binding kinetics in the P-E(2) conformation. Because Ca(2+) and H(+) binding are electrogenic reactions, fluorescent styryl dyes could be used to determine changes in the binding site occupation in equilibrium titration experiments and time-resolved relaxation processes triggered by a pH jump. By photo release from caged proton the pH of the electrolyte could be decreased in a step of 0.1 pH units by a single ultraviolet-laser flash. Analysis of the pH-jump induced relaxation process in the P-E(2) conformation showed that three Ca-ATPase-specific processes could be identified, fast H(+) binding (τ < 100 µs) and pH-insensitive conformational relaxations after the release of the Ca(2+) ion (τ â¼160 ms), and a slow process (τ â¼3.4 s) whose origin could not be unambiguously revealed. The Ca(2+)-binding affinity in the P-E(2) conformation was reduced with increasing pH, a behavior that can be explained by a reversible transition of the empty P-E(2) state to an inactivated state of the ion pump. All findings are interpreted in the framework of the Post-Albers pump cycle introduced previously, supplemented by an additional transition to an inhibited state of the ion pump.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Prótons , Retículo Sarcoplasmático/enzimologia , Animais , Sítios de Ligação , ATPases Transportadoras de Cálcio/química , Cinética , Ligação Proteica , Conformação Proteica , Coelhos , TemperaturaRESUMO
The effect of the antimycotic drug clotrimazole (CLT) on the Na,K-ATPase was investigated using fluorescence and electrical measurements. The results obtained by steady-state fluorescence experiments with the electrochromic styryl dye RH421 were combined with those achieved by a pre-steady-state method based on fast solution exchange on a solid supported membrane that adsorbs the protein. Both techniques are suitable for monitoring the electrogenic steps of the pump cycle and are in general complementary, yielding distinct kinetic information. The experiments show clearly that CLT affects specific partial reactions of the pump cycle of the Na,K-ATPase with an affinity in the low micromolar range and in a reversible manner. All results can be consistently explained by proposing the CLT-promoted formation of an ion-occluded-CLT-bound conformational E(2) state, E(2)(CLT)(X(2)) that acts as a "dead-end" side track of the pump cycle, where X stands for H+ or K+. Na+ binding, enzyme phosphorylation, and Na+ transport were not affected by CLT, and at high CLT concentrations approximately (1/3) of the enzyme remained active in the physiological transport mode. The presence of Na+ and K+ destabilized the inactivated form of the Na,K-ATPase.