Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase

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The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electrochemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time-and concentration-dependent manner consistent with a firstorder reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.Enzymes that link the transport of ions to either the hydrolysis or synthesis of ATP have been classified into three broad categories of transport ATPase, designated as P-, V-, and F-type (28). These ATPases can be distinguished by their biochemical, physical, and physiological properties. The Ftype ATPase couples the flux of ions, usually protons, down their electrochemical gradient to the synthesis of ATP. The other two classes of ATPases couple the transport of ions against their electrochemical gradient to the release of free energy from ATP hydrolysis.The mechanism by which the energy from ATP is converted into the movement of ions by the V-and P-type ATPases is poorly understood. A generalized reaction scheme has been proposed for the P-type ATPases in which transport reactions are tightly coupled to the reactions, leading to phosphorylation and dephosphorylation of the enzyme (24,28). Soon after the discovery of the V-type ATPase, it was apparent that a reaction mechLanism in which ATP hydrolysis and ion transport were tightly linked could not account for the differential inhibition and activation of the activities of

Section: Isolation Of Plasma Membranesmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Effects of Solubilization on the Inhibition of the P-Type ATPase from Maize Roots by N-(Ethoxycarbonyl)-2-Ethoxy-1,2-Dihydroquinoline

Bräuer

Gurriel²,

Tu³

1992

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“…Based on the differential inhibition of catalytic activity and proton translocation of tonoplast H+-ATPase by nitrate ( 15) and mercury (16), it has been suggested that ATP hydrolysis and proton transport are indirectly coupled. Accordingly, these inhibitors are suggested to interact with the protogenic domain of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Chilling-Induced Inactivation and Its Recovery of Tonoplast H⁺-ATPase in Mung Bean Cell Suspension Cultures

Yoshida¹

1991

“…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).…”

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confidence: 88%

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

Bräuer²,

Nungesser³

1990

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H+-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H+ leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high Hbonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.The tonoplast membrane of plant roots contains at least two enzymes, an ATPase and a pyrophosphatase, which can convert the chemical free energy released from the hydrolysis of high-energy phosphoester bonds, into a transmembrane proton, electrochemical gradient (2,6,8,33). According to the chemiosmotic concept, the resulting pH gradient and membrane potential may be used as the primary driving force to transport materials across the membrane (15,19). Indeed, the coupling of the proton gradient to the movement of Ca2+ (21,22) and sucrose (5) has been reported for tonoplast membranes. However, the molecular mechanism by which ATP hydrolysis induces vectorial proton transport remains unresolved.The relative consistency of H+/ATP ratio for the tonoplast H+-ATPase obtained under certain conditions would suggest that the coupling between ATP hydrolysis and proton pumping is direct (1). The direct coupling mechanism implies that at least one molecular event must be common to the pathways leading to ATP hydrolysis and proton pumping (14). 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,...