A FRUCTAN-FRUCTAN FRUCTOSYLTRANSFERASE ACTIVITY FROM LOLIUM-RIGIDUM

Fructan:fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidura Gaudin by a combination of af finity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose:sucrose fructosyltransf erase and invertase activities. It had an apparent pI of 4.7 as determ ined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose a nd 6(G)-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6(G)-kestose or (1,1)-kestotetraose of up to 400 mM The rate of reaction of the FFT activity was most rapid whe n assayed with 1-kestose and was less rapid when assayed with 6(G)-kes tose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity w hen assayed at a relatively high concentration of enzyme activity (app roximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerizati on > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [C-14]sucrose as subs trates, the major reaction was the transfer of a fructosyl residue fro m 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tet rasaccharide and 6(G)-kestose were also synthesized. When assayed with 6(G)-kestose and [C-14]sucrose as substrates, the major reaction of t he FFT activity was the synthesis of tetrasaccharide. However, some sy nthesis of 1-kestose and re-synthesis of 6(G)-kestose also occurred. W hen 6, kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2.7 to 3.4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan:sucros e fructosyltransferase activity of the FFT with each of the trisacchar ides, net synthesis of tetrasaccharide by the FFT was altered signific antly in the presence of sucrose. The magnitude of this effect depende d on the concentration of the trisaccharides. In the presence of sucro se, 6(G)-kestose could be a substrate of equivalent importance to 1-ke stose for synthesis of tetrasaccharide.