ModulatoryATPbinding to the E2 state of maize plasma membrane H⁺‐ATPase indicated by the kinetics of vanadate inhibition

Abstract

P-type ATPases, as major consumers of cellular ATP in eukaryotic cells, are characterized by the formation of a phosphorylated enzyme intermediate (E2P), a process that is allosterically coupled to translocation of cations against an electrochemical gradient. The catalytic cycle comprises binding of Mg-ATP at the nucleotide-binding domain, phosphorylation of the E1 state (E1), conformational transition to the E2P state, and dephosphorylation through the actuator domain and re-establishment of the E1 state. Recently, it has been suggested that, for several P-type ATPases, Mg-ATP binds to the phosphorylated enzyme, thereby accelerating the transition to the E1 state, before then becoming the enzyme's catalytic substrate. Here, we provide evidence supporting this viewpoint. We employed kinetic models based on steady-state kinetics in the presence and absence of the reversible inhibitor orthovanadate. Vanadate is generally considered to be a conformational probe that specifically binds to the E2 state, arresting the enzyme in a state analogous to the E2P state. Hydrolytic H + -ATPase activities were measured in inside-out plasma membrane vesicles isolated from roots and shoots of maize plants. For root enzymes, kinetic models of vanadate inhibition that allow simultaneous binding of Mg-ATP and vanadate to the same enzyme state were most plausible. For shoot enzymes, application of the competitive inhibitor Mg-free ATP attenuated vanadate inhibition, which is consistent with a model in which either Mg-free ATP or Mg-ATP is bound to the enzyme when vanadate binds. Therefore, data from roots and shoots indicate that binding of ATP species before transition to the E1 state plays an important role in the catalytic cycle of plant plasma membrane H + -ATPase.

The Free-Living Stage Growth Conditions of the Endophytic Fungus Serendipita indica May Regulate Its Potential as Plant Growth Promoting Microbe

“…H + transport analysis of membrane vesicles isolated from S. indica showed that preincubation with vanadate (a reversible inhibitor of these specific proton pumps - Wang et al, 2013) reduced the F max by 70%, thus confirming significant plasma membrane ATPase (P-H + -ATPase) activity in this fungus (Figure 3). This reduction in F max was independent of the C availability (2 and 20 g L −1 of glucose) under which the fungus had been grown (Figure 3C).…”

“…The addition of 20 mM NH 4 Cl was used to show a recovery of the fluorescence that indicated a collapse of the preliminarily formed H + gradient. The P-H + -ATPase proton pumping activity was measured with and without vanadate (0.2 mM Na 3 VO 4 , a reversible inhibitor – Wang et al, 2013 ) and the orthovanadate-sensitive activity was attributed to the P-H + -ATPase. Maximum fluorescence (F max ) reflects the steady-state amplitude of the ΔH formation and it was calculated as ΔF/F and expressed as percentage.…”

Characterization of Triterpene Saponin Glycyrrhizin Transport by Glycyrrhiza glabra

“…GL transport through tonoplast vesicles was dependent on MgATP and effectively inhibited by vanadate. Vanadate is a membrane ATPase inhibitor and represses the function of ABC transporters [30][31][32]. In contrast, neither niflumic acid nor probenecid affected GL transport by tonoplast vesicles.…”