Sucrose metabolism in domesticated cherry tomato, Lycopersicon esculentum var. cerasiforme Alef., and purification of sucrose synthase

Abstract

SummarySucrose metabolism was followed in developing fruit of domesticated cherry tomato. The high activities of soluble acid invertase (EC 3.2.1.26) were consistently linked with high reducing sugars, whereas Sucrose synthase (SuSy) (EC.2.4.1.13) reached a peak of activity during early stage of maturation and then decreased to near nil in mature non-growing fruits. Sucrose phosphate synthase (SPS) (EC 2.4.1.14) activity remains relatively constant throughout development, indicate it may not be an indication of cherry tomato fruit sink strength. Thus, like the large fruited types, SuSy and acid invertase, rather than SPS, could play a central role in regulating sucrose accumulation in cherry tomatoes. The result also suggested that SuSy is an indicator of sink strength in growing tomato fruit. Cultivated cherry tomato SuSy (UDP-glucose: D-fructose 2-glucosyltransferase) was purified (687-fold) 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was a homotetramer with a total molecular mass of 370 kDa and subunits of 92 kDa with maximum activity for the cleavage and synthesis of sucrose was at pH 7.0 and 8.0, respectively. The optimum temperature was 408C for both directions in HEPES-KOH buffer and an isoelectric point of 5.8. The enzymatic reaction followed typical Michaelis-Menten kinetics, with the following parameters: Km (fructose), 7.84; (UDP-glucose), 0.2612; (sucrose), 33.24; (UDP), 0.0946. The enzyme was very sensitive to inhibition by heavy metals.