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Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons

II. Properties of phosphoenolpyruvate carboxylase and cytosolic pyruvate kinase associated with the regulation of glycolysis and nitrogen assimilation

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Abstract

The cytosolic pyruvate kinase (PKC, EC 2.7.1.40) and phosphoenolpyruvate carboxylase (PEP-Case, EC 4.1.1.31) from cotyledons of 6-d-old castor seedlings (Ricinus communis L.) have been partially purified and characterized. PKC was purified 370-fold to a specific activity of 20 μmol · min 1·(mg protein)−1, and was shown to exist as a 237-kDa homotetramer. In addition, PKC displayed hyperbolic substrate saturation kinetics and demonstrated pH-dependent modulation by several metabolite effectors including glutamine, glutamate, arginine, malate and 2-oxoglutarate. Most were inhibitors at pH 6.9, while activation by glutamine, asparagine and arginine and only weak inhibition for the rest were observed at pH 7.5. PEPCase was purified 33-fold to a final specific activity of 1 μmol · min−1 · (mg protein)−1. The subunit and native Mr for the enzyme were shown to be 100 and 367 kDa, respectively, suggesting a homotetrameric native structure. PEPCase displayed a typical pH activity profile with an alkaline optimum and activity decreasing rapidly below pH 7.0. The enzyme was potently inhibited by malate, isocitrate, aspartate and glutamate at pH 7.0, whereas inhibition by these compounds was considerably diminished at pH 7.5. A model depicting the regulation of glycolytic carbon flow during amino-acid and sucrose import by castor cotyledons is proposed.

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Abbreviations

IgG:

immunoglobulin G

I50a:

inhibitor concentration producing 50‰ inhibition of enzyme activity

PKC and PKpa:

cytosolic and plastidic isoenzymes of pyruvate kinase, respectively

PEP:

phosphoenolpyruvate

PEPCase:

phosphoenolpyruvate carboxylase

3-PGA:

3-phosphoglycerate

References

  • Andreo, C.S., Gonzalez, D.H., Iglesias, A.A. (1987) Higher plant phosphoenolpyruvate carboxylase. Structure and regulation. FEBS Lett. 213, 1–8

    Google Scholar 

  • Baysdorfer, C., Bassham, J.A. (1984) Spinach pyruvate kinase isoforms. Partial purification and regulatory properties. Plant Physiol. 74, 374–379

    Google Scholar 

  • Brooks, S.P.J. (1992) A simple computer program with statistical tests for analysis of enzyme kinetics. BioTechniques 13, 906–911

    Google Scholar 

  • Cole, K.P., Blakeley, S.D., Dennis, D.T. (1992) Structure of the gene encoding potato cytosolic pyruvate kinase. Gene 122, 255–261

    Google Scholar 

  • Dixon, M., Webb, E.C. (1979) Enzymes. 3rd Ed. Academic Press, New York

    Google Scholar 

  • Duggleby, R.G., Clarke, R.B. (1991) Experimental designs for estimating the parameters of the Michaelis-Menten equation from progress curves of enzyme catalyzed reactions. Biochim. Biophys. Acta 1080, 231–236

    Google Scholar 

  • Duggleby, R.G., Dennis, D.D. (1973) The characterization and regulatory properties of pyruvate kinase from cotton seeds. Arch. Biochem. Biophys. 155, 270–277

    Google Scholar 

  • Geigenberger, P., Stitt, M. (1991) Regulation of carbon partitioning between sucrose and nitrogen assimilation in cotyledons of germinating Ricinus commuais L. seedlings. Planta 185, 563–568

    Google Scholar 

  • Hatzfeld, W-D., Stitt, M. (1991) Regulation of glycolysis in heterotrophic cell suspension cultures of Chenopodium rubrum in response to proton fluxes at the plasmalemma. Physiol. Plant. 81, 103–110

    Google Scholar 

  • Ireland, R.J., DeLuca, V., Dennis, D.T. (1980) Characterization and kinetics of isozymes of pyruvate kinase from developing castor bean endosperm. Plant Physiol. 65, 1188–1193

    Google Scholar 

  • Komor, E., Rotter, M. Tanner, W. (1977) A proton cotransport system in a higher plant: sucrose transport in Ricinus communis. Plant Sci. Lett. 9, 153–162

    Google Scholar 

  • Lin, M., Turpin, D.H., Plaxton, W.C. (1989) Pyruvate kinase isoenzymes from the green alga Selenastrum minutum. II. Kinetic and regulatory properties. Arch. Biochem. Biophys. 269, 228–238

    Google Scholar 

  • Muirhead, H. (1990) Isoenzymes of pyruvate kinase. Biochem. Soc. Trans. 18, 193–196

    Google Scholar 

  • Peterson, J.B., Evans H.J. (1978) Properties of pyruvate kinase from soybean nodule cytosol. Plant Physiol. 61, 909–914

    Google Scholar 

  • Plaxton, W.C. (1988) Purification of pyruvate kinase from germinating castor bean endosperm. Plant Physiol. 86, 1064–1069

    Google Scholar 

  • Plaxton, W.C. (1989) Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isoenzymes from castor-oil-plant endosperm and leaf. Eur. J. Biochem. 181, 443–451

    Google Scholar 

  • Plaxton, W.C., Storey, K.B. (1985) Tissue specific isozymes of pyruvate kinase in the channelled whelk Busycotypus canaliculatum: enzyme modification in response to environmental anoxia. J. Comp. Physiol. 155, 291–296

    Google Scholar 

  • Plaxton, W.C., Dennis, D.T., Knowles, V.L. (1990) Purification of leucoplast pyruvate kinase from developing castor bean endosperm. Plant Physiol. 94, 1528–1534

    Google Scholar 

  • Podestá, F.E., Andreo, C.S. (1989) Maize leaf phosphoenolpyruvate carboxylase: oligomeric structure and activity in the presence of glycerol. Plant Physiol. 90, 427–433

    Google Scholar 

  • Podestá, F.E., Plaxton, W.C. (1991) Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor beans. Biochem. J. 279, 495–501

    Google Scholar 

  • Podestá, F.E., Plaxton W.C. (1992) Plant cytosolic pyruvate kinase: a kinetic study. Biochim. Biophys. Acta 1160, 213–220

    Google Scholar 

  • Podestá, F.E., Plaxton, W.C. (1993) Activation of cytosolic pyruvate kinase by polyethylene glycol. Plant Physiol. 103, 285–288

    Google Scholar 

  • Podestá, F.E., Plaxton, W.C. (1994) Regulation of carbon metabolism in germinating Ricinus communis cotyledons. I. Developmental profiles for the activity, concentration, and molecular structure of the pyrophosphate- and ATP-dependent phosphofructokinases, phosphoercolpyruvate carboxylase and pyruvate kinase. Planta 194, 374–380

    Google Scholar 

  • Robinson, S.P., Beevers, H. (1981) Evidence for amino-acid proton cotransport in Ricinus cotyledons. Planta 152, 527–533

    Google Scholar 

  • Sangwan, R.S., Singh, N., Plaxton, W.C. (1992) Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds. Plant Physiol. 99, 445–449

    Google Scholar 

  • Schuller, K.A., Plaxton, W.C., Turpin, D.H. (1990a) Regulation of phosphoenolpyruvate carboxylase from the green alga Selenastrum minutum: properties associated with the replenishment of tricarboxylic acid cycle intermediates during ammonium assimilation. Plant Physiol. 93, 1303–1311

    Google Scholar 

  • Schuller, K.A., Turpin, D.H., Plaxton, W.C. (1990b) Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase. Plant Physiol. 94, 1429–1435

    Google Scholar 

  • Schulz, M., Klockenbring, T., Hunte, C., Schnabl, H. (1993) Involvement of ubiquitin in phosphoenolpyruvate carboxylase degradation. Bot. Acta. 106, 143–145

    Google Scholar 

  • Turpin, D.H., Botha, F.C., Smith, R.G., Feil, R., Horsey, A., Vanlerberghe, G.C. (1990) Regulation of carbon partitioning to respiration during dark ammonium assimilation in the green alga Selenastrum minutum. Plant Physiol. 96, 166–175

    Google Scholar 

  • Vanlerberghe, G.C., Schuller, K.A., Smith, R.G., Feil, R., Plaxton, W.C., Turpin, D.H. (1990) Relationship between NH +4 assimilation rate and in vivo phosphoenolpyruvate carboxylase activity: regulation of anaplerotic carbon flow in the green alga Selenastrum minutum. Plant Physiol. 94, 284–290

    Google Scholar 

  • Van Quy, L., Foyer, C., Champigny, M-L. (1991) Effect of light and NO −3 on wheat leaf phosphoenolpyruvate carboxylase activity. Plant Physiol. 97, 1476–1482

    Google Scholar 

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Author notes

  1. Florencio E. Podestá

    Present address: Centro de Estudios Fotosinteticos Y Bioquimicos, Universidad Nacional de Rosario, Suipacha 531, 2000, Rosario, Argentina

Authors and Affiliations

  1. Departments of Biology and Biochemistry, Queen's University, K7L 3N6, Kingston, Ontario, Canada

    Florencio E. Podestá & William C. Plaxton

Authors
  1. Florencio E. Podestá
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  2. William C. Plaxton
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Correspondence to William C. Plaxton.

Additional information

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

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Podestá, F.E., Plaxton, W.C. Regulation of cytosolic carbon metabolism in germinating Ricinus communis cotyledons. Planta 194, 381–387 (1994). https://doi.org/10.1007/BF00197539

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  • DOI: https://doi.org/10.1007/BF00197539

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