Partial purification and characterisation of sucrose synthase in sugarcane

Three sucrose synthase (SuSy) (EC 2.4.1.13) forms were isolated from sugarcane leaf roll tissue. During anion exchange chromatography, one peak of activity (SuSyA) eluted during the wash step and the other peak (SuSyB) during the salt gradient phase at 180mM KCl concentration. A third form of activity (SuSyC), which also eluted at 180mM KCl, was also present in the leaf roll and replaced SuSyB depending on the season of the year. Substrate Km values, as well as sucrose breakdown/synthesis ratios, differed between these forms. For SuSyA, SuSyB, and SuSyC, respectively, Km values+/-SE (mM) were: 41.8+/-3.4, 109+/-23, and 35.9+/-2.3 for sucrose, 1.07+/-0.08, 0.214+/-0.039, and 0.00191+/-0.00019 for UDP, 6.62+/-1.55, 11.7+/-2.6, and 6.49+/-0.61 for fructose, and 3.59+/-0.37, 0.530+/-0.142, and 0.234+/-0.025 for UDP-glucose. Sucrose breakdown/synthesis ratios+/-SE were 0.0791+/-0.0199, 0.330+/-0.180, and 0.426+/-0.069 for SuSyA, SuSyB, and SuSyC, respectively. The ratio of the area of peak 1 (low breakdown/synthesis ratio) to the area of peak 2 (high breakdown/synthesis ratio) in sucrose accumulating tissue (internode 9) was 0.88, while in non-accumulating (leaf roll) tissue it was 14.5 at the same time of year. The molecular mass of the denatured subunits of all three forms was 94kDa by SDS-PAGE. A polyclonal antiserum raised against SuSyB cross-reacted with all three forms on an immunoblot, but only SuSyA and SuSyB were immunoinactivated by this serum.