Kinetics of the Vacuolar H⁺-Pyrophosphatase

Abstract

The responses of the vacuolar membrane (tonoplast) protonpumping inorganic pyrophosphatase (HI-PPase) from oat (Avena sativa L.) roots to changes in Mg2" and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the be conserved as a proton gradient if the H+-PPase functions in vivo as a pump (14,22,29). Insight into the conditions under which the H+-PPase operates in vivo, and therefore into its function, would be gained if the substrate and other modulators of its activity were known.In vitro, vacuolar H+-PPases require both Mg2' and K+, in addition to PPi, for complete activity (7,20,26,27,29). However, the response of the enzyme to changes in both [PPi]0to and [Mg],,, can be complex (11,14,27,29). In a reaction medium containing PPi, Mg2", and K+, the complexes and ions present include free Mg, free PPi, MgPPi, Mg2PPi, K+, and KPPi as well as various protonated forms of the complexes (e.g. 11, 29). This makes it very difficult to test the effect of individual complexes on the activity of the H+-PPase because the concentration of any single complex cannot easily be changed without altering the concentration of some others. Nonetheless, previous kinetic studies have suggested that the enzyme is activated by both Mg2" and K+ ions and that the substrate is MgPPi (11,26,29). In addition, both free PPi and Mg2PPi might inhibit the enzyme, although this appears to depend on the tissue from which the tonoplast membranes are prepared (11,27,29).One limitation of the work reported thus far is that the validity of the conclusions drawn from kinetic experiments in vitro has not been tested by quantitative models of the observed data. This paper reports detailed descriptions of the response of the oat (Avena sativa L.) root vacuolar H+-PPase to changes in [Mg]

The Role of Magnesium, Pyrophosphate, and Their Complexes as Substrates and Activators of the Vacuolar H+-Pumping Inorganic Pyrophosphatase (Studies Using Ligand Protection from Covalent Inhibitors)

“…The results support the conclusion that Mg,PPi is the substrate and that there is a high-affinity Mgz+ binding site (Leigh et al, 1992;Baykov et al, 1993). Both sites bind their ligands with the K , values predicted by the model of Leigh et al (1992).…”

“…Goldman, University of Pennsylvania) and the stability constants listed by Leigh et al (1992). These calculations made appropriate allowance for the binding of Mg2+ to the EGTA present in the media.…”

“…Hydrolytic activity of the H+-PPase was determined as described by Leigh et al (1992), except that 10 pg mL-' L-a-lysophosphatidylcholine and 300 pg mL-l Triton X-100 were included in the assay media to disrupt vesicles and maximize hydrolytic activity. Final concentrations of MgSO, and PPi-BTP were 1.5 and 0.3 mM, respectively.…”

“…The available evidence indicates that the enzyme uses a complex of Mg2+ and PPi as its substrate, and earlier studies suggested that this was MgPPi (Walker and Leigh, 1981;Johannes and Felle, 1989;White et al, 1990). However, kinetic modeling has indicated that it may be Mg,PPi, with a K , in the range of 2 to 5 PM (Leigh et al, 1992;Rea et al, 1992a;Baykov et al, 1993). In addition, both kinetic and covalent inhibitor studies (Maeshima, 1991;Leigh et al, 1992;Baykov et al, 1993) indicate the presence of a high-affinity Mg2+ binding site with a K , in the range of 25 to 42 FM, but a second site with a K , of 0.25 to 0.46 mM was suggested by the kinetic modeling undertaken by Baykov et al (1993).…”

Isolation and Characterization of cDNAs Encoding the Vacuolar H+-Pyrophosphatase of Beta vulgaris

“…Recent accomplishments include identification (Britten et al, 1989;Rea et al, 1992a) and purification (Britten et al, 1989;Maeshima and Yoshida, 1989;Sarafian and Poole, 1989;Rea et al, 1992a) of the major substrate (MgzPPi; Leigh et al, 1992;Baykov et al, 1993) binding subunit, isolation and characterization of cDNAs encoding this subunit (Sarafian et al, 1992;Tanaka et al, 1993), and reconstitution of partially purified preparations of the enzyme to yield proteoliposomes competent in PPi-dependent H+-translocation (Britten et al, 1992).…”