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Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

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Abstract

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 μmol·(mg protein)-1·min-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Km values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg2+ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.

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Abbreviations

MW:

molecular weight

PEP (Case):

phosphoenolpyruvate (carboxylase)

SDS-PAGE:

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Centro de Estudios Fotosintéticos y Bioquímicos, Suipacha 531, 2000, Rosario, Argentina

    A. A. Iglesias, D. H. González & C. S. Andreo

Authors
  1. A. A. Iglesias
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  2. D. H. González
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  3. C. S. Andreo
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Iglesias, A.A., González, D.H. & Andreo, C.S. Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves. Planta 168, 239–244 (1986). https://doi.org/10.1007/BF00402969

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  • DOI: https://doi.org/10.1007/BF00402969

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