Isolation of sucrose: sucrose 1-fructosyltransferase (1-SST) from barley (Hordeum vulgare)

The enzyme sucrose: sucrose 1-fructosyltransferase was partially purified f rom barley leaf growth zones. Four steps (ammonium sulphate precipitation a nd polyethylene glycol precipitation, followed by chromatography on Concana valin A-sepharose and hydroxylapatite) yielded a 35-fold purification. The resulting preparation of 1-SST which still contained a number of different activities related to fructan metabolism, was subjected to preparative isoe lectric focusing, and sections of the gel were analysed individually for 1- SST and related activities, using sucrose and 1-kestose as substrates. This procedure yielded a 196-fold purification and revealed the presence of two isozymes of 1-SST with pi values of 4.93 and 4.99, as determined by analyt ical isoelectric focusing of the corresponding fractions. Both isozymes pro duced glucose and 1-kestose when incubated with sucrose. In addition, small amounts of 6-kestose and tetrasaccharides were formed. In particular, one of the two 1-SST isozymes yielded fructose when incubated with 1-kestose, i ndicating that it also acts as a fructan exohydrolase. The other isozyme ex hibited less fructan exohydrolase activity. Nystose was also degraded by th e fructan exohydrolase activity but less than 1-kestose, whereas 6-kestose was not a substrate for the enzyme. Incubation of both 1-SSTs with differen t concentrations of sucrose showed that the enzyme was not saturated even a t 500 mM. As for the barley sucrose: fructan 6-fructosyltransferase, both i sozymes of 1-SST yielded two polypeptide bands of molecular weight 50 and 2 2 kDa upon sodium dodecylsulphate polyacrylamide gel electrophoresis, sugge sting their close relationship to invertase (composed of two subunits of si milar size), as previously reported for other plants.