Substrate Kinetics of the Tonoplast H⁺-Translocating Inorganic Pyrophosphatase and Its Activation by Free Mg²⁺

Abstract

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+-

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

“…The activity of the H+-PPiase is also K+ dependent and is competitively inhibited by Na+ (White et al, 1990;Rea and Poole, 1993). This suggests that the H+-ATPase will play the principal role in energizing Na+/H+ antiport activity in cells accumulating significant quantities of NaCl.…”

Characteristics of Adenosinetriphosphatase and Inorganic Pyrophosphatase in Tonoplasts Isolated from Three CAM Species,Ananas comosus, Kalanchoë pinnataand K. daigremontiana

“…This may be related to the fact that the actual substrate of tonoplast PPase is not simply PPi, and that the free PPi is a competitive inhibitor of the tonoplast PPase (Walker and Leigh, 1981;Wang et al, 1986 ;Maeshima and Yoshida, 1989). The actual substrate of the tonoplast PPase is said to be magnesium pyrophosphate (MgPP 1 ) (Wang et al, 1986;White et al, 1990), both MgPP 1 and dimagnesium pyrophosphate (Mg 2 PP 1 ) (Walker and Leigh, 1981), or Mg 2 PP 1 alone . In contrast, the tonoplast ATPase of pineapple, K pinnata and K daigremontiana had 80 to 88% activity remaining when 3 mM MgSO 4 was replaced by 3 mM MnS0 4 and the tonoplast PPase activity of the three CAM species strictly depended on the Mg 2 + (data not shown) .…”

Effects of Bafilomycin A1 and Metabolic Inhibitors on the Maintenance of Vacuolar Acidity in Maize Root Hair Cells

“…However, our experimental conditions were not optimized to observe signals from the phosphates of ATP. The significance of changes in cytosolic Mg2+ may be of interest, because certain experiments have suggested that free Mg2+ is a positive allosteric modulator of the H+-PPase (White et al, 1990). It seems apparent that further investigations are necessary to understand the factor(s)…”