Characterization of the Effects of Divalent Cations on the Coupled Activities of the H⁺-ATPase in Tonoplast Vesicles

Abstract

The substrate requirement of the H*-ATPase in purified com root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg2' or Mn2+. The presence of Ca2+ or Ba2+ did not significantiy affect the coupled activities. The addition of Cd2', Co2+, Cu2+, and Zn2' inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu2+ and Co2+ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co2+, Cu2+, and Zn2+ were effective inhibitors. However, this inhibition could be further modulated by free Co2+, Cu2+, and Zn2 . While the equilibrium concentrations of Cd-ATP and free Cd2' were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H -ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in "indirect-link" mechanism.Based on the studies of isolated membrane vesicles, the tonoplast of plant cells has been shown to contain two inwardly directed, electrogenic H+-pumps, one powered by ATP and the other by PPi (1,2,6,14). The proton electrochemical potential, AUH+, which contains a ApH (acidic inside) and a A'I (positive inside) generated by these pumps serves as the driving force for the secondary transport of other solutes across tonoplast (3,16,17). The tonoplast H+-ATPase requires Mg-ATP as the substrate and can be inhibited by nitrate ions (7,19). The properties of this enzyme, e.g. vanadate insensitivity, anion dependence, and the generation of proton electrochemical potential, are similar to the Mg-ATPase associated with isolated vacuoles (29).Unlike the plant plasma membrane H+-ATPase, which contains only one polypeptide (molecular mass =100 kD) and forms a phosphorylated intermediate during catalysis (5,15), the tonoplast H+-ATPase is composed of at least three different peptide subunits and does not involve phosphorylation of the enzyme in the reaction pathway (4, 1 1). While the exact molecular process leading to proton pumping remains to be established, the kinetics of proton gradient generation by the H+-ATPase in tonoplast vesicles has been studied. Based on initial rate measurements, a fixed stoichiometry of 2 H+/ATP was reported for the tonoplast ATPase of red beet microsomal membranes (1). This observation is consistent with the notion that the proton pumping is direc...

Differential Inhibition of Tonoplast H+-ATPase Activities by Fluorescamine and Its Derivatives

“…The results described in this study provide further evidence to support our previous hypothesis (27,29,30) tional groups essential for the differential inhibition of proton pumping. As an example, the ineffectiveness of cyclic derivatives III illustrates that the presence of multiple H-bonding groups probably form the basis for the inhibitory nature of derivatives II.…”

“…The purity of the derivatives was established by HPTLC2 and IR movement, then Equation 5 assumes the form of F, = F, -AF*exp(-k,t) (6) As described in our previous report (30), an identical mathematical expression as Equation 6, was obtained by direct curve fitting (1 1). We have demonstrated previously (27,29) that under experimental conditions, ATP hydrolysis remained constant during the buildup of the proton gradient in corn root tonoplast vesicles.…”

“…Coupling could also be accomplished through an indirect linkage, such as a membrane Bohr effect advocated by some researchers (10, 28). In our previous work with corn root tonoplast vesicles, we demonstrated that proton pumping exhibited a greater sensitivity to nitrate inhibition (29), temperature (27), mercury (27), and divalent cations (30), as compared to ATP hydrolysis. To further explore the nature of the coupling between these two processes, we used fluorescamine modification employed in previous studies of mitochondria and purple membrane systems (13,28,31).…”

Effects of Temperature on the Coupled Activities of the Vanadate-Sensitive Proton Pump from Maize Root Microsomes

“…Recently, Inesi and de Meis (9) have proposed that the Ca transport and ATP hydrolysis by the sarcoplasmic reticulum pump are indirectly coupled. An indirect coupling mechanism has been proposed for the maize root tonoplast H+-ATPase to explain the increased sensitivity of proton transport to nitrate (18), divalent cations (19), and temperature (17) relative to ATP hydrolysis. Therefore, the reaction mechanism of the plasma membrane-type ATPases may be modeled better by an indirect coupling mechanism rather than a direct mechanism, in which transport and ATP hydrolysis share at least one step (12) as exemplified by the PostAlber cycle of the Na, K, ATPase (11) and the coupling between nucleotide displacement and reorientation of the Ca binding sites with the sacroplasmic reticulum ATPase (1).…”

Root excretion and plant tolerance to cadmium toxicity - a review

“…Such root-induced changes of rhizosphere pH play a major role in the bioavailability of many pH dependent nutrients, but also potentially toxic metals and a range of trace metals (Hinsinger et al 2006). Tu et al (1989) observed that Cd has an inhibitory action on H + excretion, and proton pump activity decreased by 60% when plants grew in the solution with 50µM Cd. Proton pump is important for providing energy in the uptake and movement of ions through plasma membrane.…”