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. 1997 Jul;114(3):901–905. doi: 10.1104/pp.114.3.901

Tris Is a Competitive Inhibitor of K+ Activation of the Vacuolar H+-Pumping Pyrophosphatase.

R Gordon-Weeks 1, V D Koren'kov 1, S H Steele 1, R A Leigh 1
PMCID: PMC158378  PMID: 12223751

Abstract

The effects of a range of commonly used pH buffers on the hydrolytic activity of the plant vacuolar H+-transporting inorganic pyrophosphatase (V-PPase) from mung bean (Vigna radiata L.) hypocotyls were tested. All of the buffers inhibited K+ stimulation of the V-PPase, and the degree of inhibition was dependent on the concentrations of both the buffer and K+. The effects were dependent on the organic cation used in the buffers, and those tested inhibited in the order: Tris > Bis-Tris-propane > Bicine = Tricine > imidazole. Detailed studies revealed that a model in which Tris affects both the Km and Vmax for K+ stimulation provided an accurate description of the observed kinetics. The ability of different cations to stimulate the V-PPase was measured with a noncompeting buffer (5 mM imidazole-HCl) and the order of effectiveness was K+ = Rb+ > NH4+ >> Cs+ > Na+ > Li+, with the Km for K+ stimulation being about 1 to 2 mM. Published experiments performed in the presence of Tris were re-evaluated and all could be fitted to mixed inhibition kinetics, with kinetic parameters similar to those measured for the mung bean V-PPase. It is concluded that the variations in the published Km for K+ stimulation of the V-PPase are probably due to the effects of pH buffer cations and that the real value for this parameter is in the low millimolar range. The implications of this for regulation of the V-PPase by K+ in vivo and for the role of the enzyme in K+ transport into the vacuole are discussed.

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Selected References

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  1. Baykov A. A., Bakuleva N. P., Rea P. A. Steady-state kinetics of substrate hydrolysis by vacuolar H(+)-pyrophosphatase. A simple three-state model. Eur J Biochem. 1993 Oct 15;217(2):755–762. doi: 10.1111/j.1432-1033.1993.tb18303.x. [DOI] [PubMed] [Google Scholar]
  2. Baykov A. A., Sergina N. V., Evtushenko O. A., Dubnova E. B. Kinetic characterization of the hydrolytic activity of the H+-pyrophosphatase of Rhodospirillum rubrum in membrane-bound and isolated states. Eur J Biochem. 1996 Feb 15;236(1):121–127. doi: 10.1111/j.1432-1033.1996.00121.x. [DOI] [PubMed] [Google Scholar]
  3. Davies J. M., Rea P. A., Sanders D. Vacuolar proton-pumping pyrophosphatase in Beta vulgaris shows vectorial activation by potassium. FEBS Lett. 1991 Jan 14;278(1):66–68. doi: 10.1016/0014-5793(91)80085-h. [DOI] [PubMed] [Google Scholar]
  4. Gordon-Weeks R., Steele S. H., Leigh R. A. 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). Plant Physiol. 1996 May;111(1):195–202. doi: 10.1104/pp.111.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kim E. J., Zhen R. G., Rea P. A. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6128–6132. doi: 10.1073/pnas.91.13.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kim Y., Kim E. J., Rea P. A. Isolation and characterization of cDNAs encoding the vacuolar H(+)-pyrophosphatase of Beta vulgaris. Plant Physiol. 1994 Sep;106(1):375–382. doi: 10.1104/pp.106.1.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leigh R. A., Gordon-Weeks R., Steele S. H., Koren'kov V. D. The H(+)-pumping inorganic pyrophosphatase of the vacuolar membrane of higher plants. Symp Soc Exp Biol. 1994;48:61–75. [PubMed] [Google Scholar]
  8. Leigh R. A., Pope A. J., Jennings I. R., Sanders D. Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors. Plant Physiol. 1992 Dec;100(4):1698–1705. doi: 10.1104/pp.100.4.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Rea P. A., Poole R. J. Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles. Plant Physiol. 1985 Jan;77(1):46–52. doi: 10.1104/pp.77.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sakakibara Y., Kobayashi H., Kasamo K. Isolation and characterization of cDNAs encoding vacuolar H(+)-pyrophosphatase isoforms from rice (Oryza sativa L.). Plant Mol Biol. 1996 Aug;31(5):1029–1038. doi: 10.1007/BF00040721. [DOI] [PubMed] [Google Scholar]
  11. Sarafian V., Kim Y., Poole R. J., Rea P. A. Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1775–1779. doi: 10.1073/pnas.89.5.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sato M. H., Kasahara M., Ishii N., Homareda H., Matsui H., Yoshida M. Purified vacuolar inorganic pyrophosphatase consisting of a 75-kDa polypeptide can pump H+ into reconstituted proteoliposomes. J Biol Chem. 1994 Mar 4;269(9):6725–6728. [PubMed] [Google Scholar]
  13. Sze H., Ward J. M., Lai S. Vacuolar H(+)-translocating ATPases from plants: structure, function, and isoforms. J Bioenerg Biomembr. 1992 Aug;24(4):371–381. doi: 10.1007/BF00762530. [DOI] [PubMed] [Google Scholar]
  14. Tanaka Y., Chiba K., Maeda M., Maeshima M. Molecular cloning of cDNA for vacuolar membrane proton-translocating inorganic pyrophosphatase in Hordeum vulgare. Biochem Biophys Res Commun. 1993 Feb 15;190(3):1110–1114. doi: 10.1006/bbrc.1993.1164. [DOI] [PubMed] [Google Scholar]
  15. Taussig R., Gilman A. G. Mammalian membrane-bound adenylyl cyclases. J Biol Chem. 1995 Jan 6;270(1):1–4. doi: 10.1074/jbc.270.1.1. [DOI] [PubMed] [Google Scholar]
  16. Walker D. J., Leigh R. A., Miller A. J. Potassium homeostasis in vacuolate plant cells. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10510–10514. doi: 10.1073/pnas.93.19.10510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wang Y., Leigh R. A., Kaestner K. H., Sze H. Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots. Plant Physiol. 1986 Jun;81(2):497–502. doi: 10.1104/pp.81.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. White P. J., Marshall J., Smith J. A. Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg. Plant Physiol. 1990 Jul;93(3):1063–1070. doi: 10.1104/pp.93.3.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]

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