TAILIEUCHUNG - Báo cáo Y học: Kinetic properties of bifunctional 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase from spinach leaves

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. . | Eur. J. Biochem. 269 1267-1277 2002 FEBS 2002 Kinetic properties of bifunctional 6-phosphofructo-2-kinase fructose-2 6-bisphosphatase from spinach leaves Jonathan E. Markham and Nicholas J. Kruger Department of Plant Sciences University of Oxford South Parks Road Oxford OX1 3RB UK A cDNA encoding 6-phosphofructo-2-kinase fructose-2 6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa estimated by gel filtration chromatography and a subunit size of kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity nmol-min-1-mg protein-1 and fructose-2 6-bisphosphatase specific activity nmol-min-1-mg protein-1 . The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2 6-bisphosphatase activity was sensitive to product inhibition inhibition by inorganic phosphate was uncompetitive whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2 6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2 6-bisphosphatase activities of the bifunctional enzyme. Keywords fructose 2 6-bisphosphate .

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