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Vanadate-sensitive ATPase in the plasmalemma ofAcetabularia: biochemical and kinetic characterization

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Abstract

The electrogenic Cl−-pump in the plasmalemma of the marine algaAcetabularia acetabulum (L.) Silva has been suggested to be an unusual type of ATPase (Gradmann, 1989, Methods Enzymol.174, 490). For a biochemical treatment of this issue, a plasmalemma-rich membrane fraction fromAcetabularia has been prepared by phase-partitioning. About 80% of the ATPase activity in this material is inhibited by vanadate (K I50=1–2 μM). The phosphohydrolytic properties of the corresponding enzyme were further investigated. Its primary substrate MgATP2− (K m about 270 μM). Compared with other plasmalemma ATPases, it has an extremely alkaline pH optimum (pH 8–8.5), a weak sensitivity to diethyl-stilbestrol and to N,N'-dicyclohexylcarbodiimide, a strong selectivity for Mg2+ over alternative divalent cations, and a weak selectivity for ATP over other phosphohydrolytic substrates. It is insensitive to KCl at concentrations up to 200 mM. Both ATP4− and Mg2ATP inhibit the ATPase, satisfying a relationship for competitive inhibition by 2ATP4− (K IATP=1.56 mM) and noncompetitive inhibition by Mg2ATP (K IMg2ATP=1.35 mM). Since no transport experiments are reported in this study, the ion species (H+ or Cl−) that is transferred by this ATPase is not identified.

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Abbreviations

Bis-tris-propane:

1,3-bis[tris(hydroxymethyl)methylaminopropane

DCCD:

N,N'-dicyclohexylcarbodiimide

DES:

diethylstilbestrol

DOC:

sodium deoxycholate

Mes:

2-(N-morpholino)ethanesulfonic acid

PCMBS:

p-chloromercuri-benzenesulfonate

PM:

plasmalemma-enriched membrane fraction

References

  • Albertsson, P.A. (1958) Particle fractionation in liquid two-phase systems. Biochim. Biophys. Acta27, 378–395

    PubMed  Google Scholar 

  • Anthon, G.E., Spanswick, R.M. (1986) Purification and properties of the H+-translocating ATPase from the plasma membrane of tomato roots. Plant Physiol.81, 1080–1085

    Google Scholar 

  • Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase inBeta vulgaris. Plant Physiol.24, 1–15

    Google Scholar 

  • Bennet, A.B., O'Neill, S.D., Eilman, M., Spanswick, R.M. (1985) H+-ATPase activity from storage tissue ofBeta vulgaris. Plant Physiol.79, 495–499

    Google Scholar 

  • Blair, J.McD. (1970) Magnesium, potassium, and the adenylate kinase equilibrium. Eur. J. Biochem.13, 384–390

    PubMed  Google Scholar 

  • Blum, H., Beier, H., Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis8, 93–99

    Google Scholar 

  • Bowman, B.J., Bowman, E.J. (1986) H+-ATPases from mitochondria, plasma membranes, and vacuoles of fungal cells. J. Membr. Biol.94, 83–97

    PubMed  Google Scholar 

  • Brüggemann, W., Janiesch, P. (1987) Characterization of plasma membrane H+-ATPase from salt-tolerant and salt-sensitivePlantago species. J. Plant Physiol.130, 395–411

    Google Scholar 

  • Clement, J.-D., Blein, J.-P., Rigaud, J., Scalla, R. (1986) Characterization of ATPase from maize shoot plasma membrane prepared by partition in an aqueous polymer two phase system. Physiol. Veg.24, 25–35

    Google Scholar 

  • DuPont, F.M., Burke, L.L., Spanswick, R.M. (1981) Characterization of a partially purified adenosine triphosphatase from corn root plasma membrane fractions. Plant Physiol.67, 59–63

    Google Scholar 

  • Gerencser, G.A., White, J.F., Gradmann, D., Bonting, S. (1988) Is there a Cl− pump? Am. J. Physiol.255, R677-R692

    PubMed  Google Scholar 

  • Goldfarb, V., Gradmann, D. (1983) ATPase activities in partially purified membranes ofAcetabularia. Plant Cell Rep.2, 152–155

    Google Scholar 

  • Goldfarb, V., Sanders, D., Gradmann, D. (1984) Reversal of electrogenic Cl− pump inAcetabularia increases level and32P labelling of ATP. J. Exp. Bot.35, 645–658

    Google Scholar 

  • Gradmann, D. (1989) ATP-driven chloride pump in giant algaAcetabularia. Methods Enzymol.174, 490–504

    Google Scholar 

  • Graves, J.S., Gutknecht, J. (1977a) Chloride transport and the membrane potential in the marine algaHalicystis parvula. J. Membr. Biol.36, 65–81

    Google Scholar 

  • Graves, J.S., Gutknecht, J. (1977b) Current-voltage relationships and voltage sensitivity of the Cl− pump inHalicystis parvula. J. Membr. Biol.36, 83–95

    Google Scholar 

  • Hämmerling, J. (1963) Nucleo-cytoplasmic interactions inAcetabularia and other cells. Annu. Rev. Plant Physiol.14, 65–92

    Google Scholar 

  • Hexum, T., Samson, F.E., Himes, R.H. (1970) Kinetic studies of membrane (Na+-K+-Mg2+)-ATPase. Biochim. Biophys. Acta212, 322–331

    PubMed  Google Scholar 

  • Hill, B.S., Hill, A.E. (1973) ATP-driven chloride-ATPase fromLimonium salt gland. J. Membr. Biol.12, 147–158

    Google Scholar 

  • Hodges, T.K., Mills, D. (1986) Isolation of the plasma membrane. Methods Enzymol.118, 41–54

    Google Scholar 

  • Huang, L., Berry, E.A. (1990) Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue. Biochim. Biophys. Acta1039, 241–252

    PubMed  Google Scholar 

  • Ikeda, M., Oesterhelt, D. (1990) A Cl−-translocating adenosinetriphosphatase inAcetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution. Biochemistry29, 2065–2070

    PubMed  Google Scholar 

  • Ikeda, M., Schmid, R., Oesterhelt, D. (1990) A Cl−-translocating adenosinetriphosphatase inAcetabularia acetabulum. 1. Purification and characterization of a novel type of adenosinetriphosphatase that differs from chloroplast F1 adenosinetriphos-phatase. Biochemistry29, 2057–2065

    PubMed  Google Scholar 

  • Kasai, M., Muto, S. (1990) Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves. J. Membr. Biol.114, 133–142

    PubMed  Google Scholar 

  • Kasamo, K. (1987) Reconstitution and characterization of H+-translocating ATPase from the plasma membrane ofPhaseolus mungo L. roots. Plant Cell Physiol.28, 19–28

    Google Scholar 

  • Koland, J.G., Hammes, G.G. (1986) Steady state kinetic studies of purified yeast plasma membrane proton-translocating ATPase. J. Biol. Chem.261, 5936–5942

    PubMed  Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685

    PubMed  Google Scholar 

  • Lanzetta, P.A., Alvarez, L.J., Reinach, P.S., Candia, O.A. (1979) An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem.100, 95–97

    PubMed  Google Scholar 

  • Miwa, T., Iriki, Y., Suzuki, T. (1961) Mannan and xylan as essential cell wall constituents of some siphonous green algae. Coll. Int. Centre Nat. Rech. Sci.103, 113–144

    Google Scholar 

  • Nagao, T., Sasakawa, H., Sugiyama, T. (1987) Purification of a H+-ATPase from the plasma membrane of maize roots and preparation of its antibody. Plant Cell Physiol.28, 1181–1186

    Google Scholar 

  • Noat, G., Ricard, J., Borel, M., Got, C. (1970) Kinetic study of yeast hexokinase: Inhibition of the reaction by magnesium and ATP. Eur. J. Biochem.13, 347–363

    PubMed  Google Scholar 

  • O'Neill, S.D., Spanswick, R.M. (1984) Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris. J. Membr. Biol.79, 245–256

    Google Scholar 

  • O'Sullivan, W.J., Perrin, D.D. (1964) The stability constants of metal-adenine nucleotide complexes. Biochemistry3, 18–26

    Google Scholar 

  • Parr, A.J., Hanke, D.E. (1982) ‘Chloride-stimulated ATPase’ activity inLimonium vulgare Mill. Phil. Trans. R. Soc. Lond.B299, 459–468

    Google Scholar 

  • Pedersen, P.L., Carafoli, E. (1987) Ion motive ATPases. I. Ubiquity, properties, and significance to cell function. TIBS12, 146–150

    Google Scholar 

  • Peterson, G.L. (1977) A simplification of the protein assay method of LOWRY et al. which is more generally applicable. Anal. Biochem.83, 346–356

    PubMed  Google Scholar 

  • Raven, J.A. (1989) Transport systems in algae and bryophytes: An overview. Methods Enzymol.174, 366–390

    Google Scholar 

  • Robinson, J.D. (1974) Nucleotide and divalent cation interactions with the (Na+-K+)-dependent ATPase. Biochim. Biophys. Acta341, 232–247

    PubMed  Google Scholar 

  • Saddler, H.D.W. (1970) The ionic relations ofAcetabularia mediterranea. J. Exp. Bot.21, 345–359

    Google Scholar 

  • Schmid, R., Gieseke, R. (1985) Large scale culture ofAcetabularia. In:Acetabularia 1984, pp. 319–325, Bonotto, S., Cinelli, F., Billiau, R., eds. Belgian Nuclear Center, Mol

    Google Scholar 

  • Schweiger, H.-G., Dehm, P., Berger, S. (1977) Culture conditions forAcetabularia. In: Progress inAcetabularia research, pp. 319–330, Woodcock, C.L.F., ed. Academic press, New York

    Google Scholar 

  • Sekler, I., Gläser, H.-U., Pick, U. (1991) Characterization of a plasma membrane H+-ATPase from the extremely acidophilic algaDunaliella acidophila. J. Membr. Biol.121, 51–57

    PubMed  Google Scholar 

  • Serrano, R. (1984) Plasma membrane ATPase of fungi and plants as a novel type of proton pump. Curr. Top. Cell. Regul.23, 87–126

    PubMed  Google Scholar 

  • Sillén, L.G., Martell, A.E. (1971) Stability constants of metal-ion complexes. The Chemical Society, Special Publications 25, Suppl. No. 1., London

  • Smahel, M. (1990) Spezielle ATPasen ausAcetabularia. Ph. D. Thesis University Göttingen, FRG

    Google Scholar 

  • Smahel, M., Hamann, A., Gradmann, D. (1990) The prime plasmalemma ATPase of the halophilic algaDunaliella bioculata: Purification and characterization. Planta181, 496–504

    Google Scholar 

  • Spanswick, R.M. (1989) Vacuolar and cell membrane H+-ATPases of plant cells. Ann. N. Y. Acad. Sci.574, 180–188

    PubMed  Google Scholar 

  • Storer, A.C., Cornish-Bowden, A. (1976) Concentration of MgATP2− and other ions in solution: Calculation of the true concentrations of species present in mixtures of associating ions. Biochem. J.159, 1–5

    PubMed  Google Scholar 

  • Sze, H. (1984) H+-translocating ATPases of the plasma membrane and tonoplast of plant cells. Physiol. Plant.61, 683–691

    Google Scholar 

  • Sze, H. (1985) H+-translocating ATPases: Advances using membrane vesicles. Annu. Rev. Plant Physiol.36, 175–208

    Google Scholar 

  • Thom, M., Komor, E. (1984a) Role of the ATPase of sugar-cane vacuoles in energization of the tonoplast. Eur. J. Biochem.138, 93–99

    PubMed  Google Scholar 

  • Thom, M., Komor, E. (1984b) Effect of magnesium and ATP on ATPase of sugarcane vacuoles. Planta161, 361–365

    Google Scholar 

  • Vara, F., Serrano, R. (1982) Partial purification and properties of the proton-translocating ATPase of plant plasma membranes. J. Biol. Chem.257, 12826–12830

    PubMed  Google Scholar 

  • Widell, S., Lundborg, T., Larsson, C. (1982) Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system. Plant Physiol.70, 1429–1435

    Google Scholar 

  • Williams, L.E., Hall, J.L. (1989) ATPase and proton translocating activities in a plasma membrane-enriched fraction from cotyledons ofRicinus communis. J. Exp. Bot.40, 1205–1213

    Google Scholar 

  • Yoshida, S., Uemura, M., Niki, T., Sakai, A., Gusta, L.V. (1983) Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants. Plant Physiol.72, 105–114

    Google Scholar 

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Author notes

  1. Hans-Georg Klieber

    Present address: Physiologisches Institut der Technischen Universität, Biedersteinerstrasse 29, W-8000, 40, München, FRG

Authors and Affiliations

  1. Pflanzenphysiologisches Institut der Universität, Untere Karspüle 2, W-3400, Göttingen, Germany

    Martin Smahel, Hans-Georg Klieber & Dietrich Gradmann

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  1. Martin Smahel
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  2. Hans-Georg Klieber
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  3. Dietrich Gradmann
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Additional information

This work was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. Hanna Bork for culturingAcetabularia and Dr. Gerhard Thiel for critical reading of the manuscript.

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Smahel, M., Klieber, HG. & Gradmann, D. Vanadate-sensitive ATPase in the plasmalemma ofAcetabularia: biochemical and kinetic characterization. Planta 188, 62–69 (1992). https://doi.org/10.1007/BF01160713

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  • DOI: https://doi.org/10.1007/BF01160713

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