To clarify the kinetic characteristics and ionic requirements of the tonoplast H+-translocating inorganic pyrophosphatase (H+-PPiase), PPi hydrolysis and PPi-dependent H+ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoe Solute accumulation in plant vacuoles appears to be energized by two distinct H+-translocating enzymes at the tonoplast, an H+-ATPase and an H+-PPiases (11, 25). The tonoplast H+-ATPase has been studied extensively, and its kinetic characteristics are relatively well established (1,25,30). In contrast, the kinetics of the H+-PPiase are not yet completely understood, mainly because of the difficulty of varying independently the concentrations of potential substrates and effectors when assaying this enzyme. However, a more complete kinetic description of the tonoplast H+-PPiase would be useful in elucidating the physiological role of this enzyme and in explaining how its activity might be modulated in vivo (17,25,31).Of the properties described thus far for the tonoplast H+-PPiase, there is general agreement that the enzyme is stimulated greatly by K+ and, to a lesser extent, by other alkalimetal cations (19,24,34,35). H+-PPiase activity is also Mgdependent, and when assayed at fixed PPi concentrations usually exhibits a sigmoidal response to increasing total Mg concentrations (5,32,34,35). On the other hand, in the presence of saturating Mg, the activity of the H+-PPiase typically shows hyperbolic kinetics with respect to total PPi concentration, with an apparent Km for total PPi between 5 and 30 ,M (5, 10, 13, 19, 20, 23 24, 35). These observations suggest that an Mg/PPi complex is the true substrate for the enzyme, and both 34,35) and Mg2PPi (34) have been proposed as possible substrates. In fact, the substrate kinetics of the tonoplast H+-PPiase have been difficult to interpret unambiguously, and there appear to be species differences in the effects ofsupraoptimal Mg/PPi concentrations, which markedly reduce PPiase activity in some preparations (4,10,13,17,32,35), but not in others (5,19,34).By analogy with the cytoplasmic PPiases ofmicroorganisms (7,14), it has also been suggested that the activity of the tonoplast H+-PPiase is strongly modulated by certain effectors. In particular, free Mg2' has been proposed as an activator of the enzyme (13,34,35) and free PPi (i.e. PPi4-, or H2PPi2-) as a competitive inhibitor (34,35), with Mg2PPi also possibly being inhibitory at high concentrations (13). However, a complete kinetic description of the tonoplast H+-
