Allosteric regulation by Mg2+ of the vacuolar H(+)-PPase from Acer pseudoplatanus cells. Ca2+/Mg2+ interactions.
The tonoplast H(+)-PPase was previously characterized in Acer pseudoplatanus cells (Pugin et al (1991) Plant Sci 73, 23-34; Fraichard et al (1993) Plant Physiol Biochem 31, 349-359). Tonoplast vesicles were obtained from vacuoles isolated from protoplasts of A pseudoplatanus suspension cultures and used to study kinetic effects of Mg2+ and Ca2+ on PPi hydrolysis. The concentrations of ionic species (free Mg2+, free PPi, and MgPPi complexes) were calculated with apparent dissociation constants of 55.3 microM for MgPPi and 59.6 microM for CaPPi. Our results indicated that the substrate of the tonoplast PPase was a MgPPi complex and that free Mg2+ was essential for PPi hydrolysis. With fixed free Mg2+ concentrations, PPase activity showed Michaelis-Menten kinetics with respect to MgPPi. Moreover, free Mg2+ acted as an allosteric activator with a Hill coefficient of 2.4, indicating at least two Mg2+ binding sites on the enzyme. The Mg-imidodiphosphate complex was a competitive inhibitor of the substrate MgPPi but did not change significantly the allosteric activation by free Mg2+. This result confirmed the presence of Mg2+ regulatory sites. Ca2+ acted as an uncompetitive inhibitor of MgPPi hydrolysis. Furthermore, the sensitivity of the H(+)-PPase to Ca2+ increased with decrease in free Mg2+ concentration. Therefore, Ca2+ and Mg2+ may compete for a common binding site. Taken together, our results confirm that activation by free Mg2+ and inhibition by Ca2+ could be involved in the regulation of the PPase activity in vivo.
Fraichard
A
A
Institut de Biologie et de Physiologie Végétales, Université de Lausanne, Switzerland.
Trossat
C
C
Perotti
E
E
Pugin
A
A
D000494:Allosteric Regulation; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014197:Trees / Q000166:cytology / Q000201:enzymology*; D014617:Vacuoles / Q000201:enzymology*
D009943:Organophosphorus Compounds; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D002118:Calcium