Purification and properties of sucrose synthetase from morning-glory callus cells

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Abstract
Sucrose synthetase was purified about 130-fold from morning-glory (Pharbitis nil Choisy cv. Murasaki) callus cells, and the properties of sucrose synthesis and cleavage activities of the enzyme were compared. The enzyme preparation gave a single band by disc electrophoresis. The molecular mass of the enzyme was estimated to be 4.2 × 105 by gel filtration. The enzyme preparation gave two bands by SDS disc electrophoresis, suggesting the molecular mass of about 3.8 ×104 and 7.0 × 104. The pH optima of sucrose synthesis and cleavage activities of the enzyme were different from each other, giving pH 9.0 and pH 6.5 respectively. MgCl2, MnCl2 and CaCl2 activated the sucrose synthesis activity about two times the normal rate and conversely inhibited the sucrose cleavage activity. F-6-P was not replaced by fructose. UDP was the only valuable substrate as a nucleotide diphosphate. The enzyme showed the negative ecoperativity effect of UDPG suggesting to be an allosteric enzyme. The Km values of sucrose and fructose were calculated to be 167 mM and 5 mM, respectively. UDP suggested substrate inhibition. The apparent equilibrium constant varied between 1 to 3. Based on these results, the role of the enzyme in the sucrose metabolism of morning-glory callus cells will be discussed.