Sucrose phosphate synthetase : Separation from sucrose synthetase and a study of its properties

A method for the complete separation of sucrose phosphate synthetase (EC 2.4.1.14) and sucrose synthetase (EC 2.4.1.13) from wheat (Triticum aestivum L.) germ is described. The separation is achieved by chromatography on DEAE-cellulose at pH 6.5. The sucrose phosphate synthetase obtained can be further purified by gel filtration. Disc electrophoresis of sucrose phosphate preparations reveals the presence of isoenzymes. Molecular weight estimates of sucrose phosphate synthetase by gel filtration and sedimentation velocity give a value of 380,000. The enzyme is inhibited by various anions, particularly citrate, maleate, and phosphate. Activity estimate should be carried out with Good's buffers in order to avoid inhibition. Nucleoside triphosphates are competitive inhibitors toward UDP-glucose. The enzyme is sensitive to sulfhydryl reagents, but activity can be restored with DTT or β-mercapto ethanol. The fact that the enzyme is inhibited by δ-gluconolactone suggests that the reaction occurs through the formation of an unstable glucose-enzyme complex. Mg(2+) can restore enzyme activity to control values when inhibited by nucleoside triphosphates, citrate, or phosphate.