Sucrose Metabolism and Purification and Characterization of Sucrose Synthase from Lycopersicon esculentum var. cerasiforme Fruit

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  • 1 Dept. of Agroindustrial Sciences, Faculty of Agriculture, Kagawa Univ., Miki, Ikenobe 2393, Kagawa 761-07, Japan

Sucrose metabolism was followed in developing fruit of domesticated cherry tomato (Lycopersicon esculentum var. cerasiforme Alef.). The high amounts of reducing sugars were consistently linked to high soluble acid invertase (EC 3.2.1.26), whereas sucrose synthase (EC 2.4.1.13) followed the same pattern of sucrose levels and reached a peak of activity during early stage of maturation and then decreased to near nil. In comparison, sucrose phosphate synthase (EC 2.4.1.14) activity remain relatively constant throughout development. Thus, sucrose synthase and acid invertase, rather than sucrose phosphate synthase, are the critical enzymes regulating sucrose accumulation in tomatoes. Cultivated cherry tomato sucrose synthase (UDP-glucose: D-fructose 2-glucosyltransferase) was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography on DEAE-Toyopreal 650, and gel filtration on Sephadex G-200. Further purification to homogeneity resulted from a single band from SDS-PAGE. The enzyme was identified as a homotetramer with a total molecular mass of 370 kDa and subunits of 92 kDa. The enzyme showed maximum activity for the cleavage and synthesis of sucrose was at pH 7.0 and 8.0, respectively, and the optimum temperature was 40°C in both directions for HEPES-KOH buffer. The enzymatic reaction followed typical Michaelis–Menten kinetics, with the following parameters: Km (fructose),7.4; Km (UDP-glucose), 0.2612; Km (sucrose), 33.24; Km (UDP), 0.0946. The enzyme was very sensitive to inhibition by heavy metals.

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