EurekaMag.com logo
+ Site Statistics
References:
53,869,633
Abstracts:
29,686,251
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on LinkedInFollow on LinkedIn

+ Translate

Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation



Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation



European Journal of Biochemistry 267(14): 4465-4476



Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.

(PDF emailed within 0-6 h: $19.90)

Accession: 003538283

Download citation: RISBibTeXText

PMID: 10880970

DOI: 10.1046/j.1432-1327.2000.01495.x



Related references

Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures: implications for the integration of glycolysis with nitrogen assimilation. European Journal of Biochemistry 267(14): 4477-4485, 2000

Evolution of phosphoenolpyruvate carboxylase activity and lipid content during seed maturation of two spring rapeseed cultivars (Brassica napus L.). Comptes Rendus Biologies 329(9): 719-725, 2006

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. Planta 194(3): 381-387, 1994

Expression Profiles of Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase Genes in Phalaenopsis, Implications for Regulating the Performance of Crassulacean Acid Metabolism. Frontiers in Plant Science 9: 1587-1587, 2018

Characterization and expression analysis of genes encoding phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase of Lotus japonicus, a model legume. Molecular Plant-Microbe Interactions 16(4): 281-288, 2003

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity. Planta 00(2): 174-180, 1996

Phosphate starvation and a glycolytic bypass catalyzed by phosphoenolpyruvate carboxylase in suspension-cultured Catharanthus roseus cells. Zeitschrift fuer Naturforschung Section C Biosciences 49(11-12): 742-750, 1994

Purification and characterization of phosphoenolpyruvate carboxylase from developing seeds of Brassica. Journal of Plant Biochemistry & Biotechnology 4(1): 11-16, 1995

Upregulation of vacuolar H+-translocating pyrophosphatase by phosphate starvation of Brassica napus (rapeseed) suspension cell cultures. FEBS Letters 486(2): 155-158, 2000

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism. Plant Physiology 135(1): 587-598, 2004

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of Sorghum vulgare. Planta 225(4): 801-812, 2007

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of Sorghum vulgare. Planta 225(4): 801-812, 2006

Regulatory phosphorylation of banana fruit phosphoenolpyruvate carboxylase by a copurifying phosphoenolpyruvate carboxylase-kinase. European Journal of Biochemistry 247(2): 642-651, 1997

A conserved 19-amino acid synthetic peptide from the carboxy terminus of phosphoenolpyruvate carboxylase inhibits the in vitro phosphorylation of the enzyme by the calcium-independent phosphoenolpyruvate carboxylase kinase. Plant Physiology 132(2): 1097-1106, 2003

Phosphoenolpyruvate carboxylase activity in relation to physiological processes during the growth of cell suspension cultures from Nicotiana tabacum. Physiologie Vegetale 21(5): 1031-1039, 1983