Regulation of Vacuolar H⁺-Pyrophosphatase by Free Calcium

Abstract

The HW-translocating inorganic pyrophosphatase (H -PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2" and a number of other divalent cations (Co2, Mn2", Zn2+ ATPase2 (EC 3.6.1.3) and a H+-PPase (EC 3.6.1.1) (28). The two pumps have been purified to near-homogeneity by independent techniques (6,18,25,29).It is now clear that both the vacuolar H+-ATPase and H+-PPase mediate electrogenic H+ translocation into the same intracellular compartment. Individual mechanically isolated vacuoles display both ATP-and PPi-dependent inward currents (12), and the steady-state transmembrane pH difference and electrical potential difference generated by the ATPase and PPase in isolated vacuolar vesicles interact nonadditively and are subject to kinetic control by a common H+-electrochemical potential difference (14,28). The continuous and simultaneous operation of both pumps in vivo might, however, appear redundant. Therefore, it is important to know whether the two enzymes are under independent cytoplasmic control. This question is relevant not only for tonoplast energization, but also for gaining a deeper understanding of the general metabolic role of PPi.PPi is becoming increasingly recognized as an important metabolite in plant cells, where it is present in the cytosol at concentrations of between 200 and 300 Mm (32, 34). Consideration of two of the best-characterized (soluble) PPi-dependent enzymes in plants-UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase-indicates that modulation of cellular PPi levels could have profound metabolic consequences. Thus, the cytosolic reactions catalyzed by UDP-glucose pyrophosphatase and PPi fructose-6-P-1-phosphotransferase are close to equilibrium in physiological conditions (34). Diminished cytosolic PPi would therefore be expected to favor the formation of fructose-6-P from fructoseThe vacuolar membrane (tonoplast) of higher plant cells contains two primary H+ pumps: a vacuolar-type H+-

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 in this paper support the conclusion that Mg,PPi is the substrate of the H+-PPase, as suggested by kinetic modeling of the response of the enzyme to changes in Mg2+ and PPi concentrations (Leigh et al, 1992;Rea et al, 1992a;Baykov et al, 1993). This complex protects the H+-PPase from inhibition by both NEM and PGO, whereas high concentrations of MgPPi provide no protection, and protection increases as the concentration of MgPPi decreases (Fig.…”

“…The standard preincubation mixture (total volume 50 pL) contained inhibitor, 20 mM Hepes-BTP, pH 8.0, and 3 mM EGTA (to ensure that there was no free Ca2+ in the medium; Rea et al, 1992a). When mixtures of MgSO, and PPi-BTP were included in the preincubation medium, the total concentrations needed to give the required concentrations of free Mg", MgPPi, or Mg,PPi were calculated using the computer program SOLCON (written by D.C.S.…”

“…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).…”

Gibberellic Acid Induces Vacuolar Acidification in Barley Aleurone.

“…Because GA3 increases cytoslic Ca2+ in cereal aleurone cells from -50 to 200 nM to -300 to 600 nM (Bush and Jones, 1988;Gilroy and Jones, 1992;Bush, 1996), it seems unlikely that V-PPase proton pumping would contribute to GAB-stimulated vacuole acidification. The inhibitory effects of Ca2+ on V-PPase activity are markedly reduced by Mg2+ concentrations >1 mM (Rea et al, 1992). Because aleurone vacuoles contain substantial Mg2+ reserves (Stewart et al, 1988) that are released to the cell exterior via the cytosol (Jones, 1973), regulation of the V-PPase by cations in the aleurone cell is likely to be complex and will require information on the dynamics of Mg2+ concentration in the cytosol during response to GA3.…”

Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate

“…Fourth, agents which are known to inhibit substrate hydrolysis by the V-PPase inhibit oxygen exchange. Potassium fluoride and Ca 2+, both of which are potent inhibitors of the V-PPase [18][19][20], inhibit medium P~-HOH oxygen exchange by more than 90% whereas addition of the type-specific V-ATPase "inhibitor bafilomycin A~ [21] causes 50% stimulation (Table 1). Likewise, pretreatment of vesicles with N-ethylmaleimide in the presence of both Mg 2+ and PPi [3], which irreversibly inhibits only 6% of the total PPi hydrolytic activity but 65% of the total ATP hydrolytic activity, diminishes the rate of exchange by only 27% when the F-ATPase inhibitor, NaN 3, is included in the reaction medium (Table 1).…”