Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan‐polymerizing enzyme preparation

Abstract

A partially purified enzyme preparation from leaves of Lolium temulentum L. was previously shown to catalyse the net synthesis of oligofructans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magnitude and properties of these hydrolytic activities have been determined. The significance of these catabolic activities for studies of fructan polymerization both in vitro and in tissues in a physiologically anabolic state are discussed.The preparation hydrolysed 1-kestose, 6-kestose, neokestose, inulin oligosaccharides of low degree of polymerization (DP 4 and 5) and endogenous oligofructans from L. temulentum, with the concomitant release of monosaccharide. The preparation also released reducing sugar at low rates from high molecular weight inulin but had no detectable activity against bacterial levan. Simultaneous incubation of sucrose and Neosugar (a commercially available mixture of predominantly β-2, 1 linked tri-, tetra-and penta-saccharides) showed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentrations 30 mol m −$ . The kinetic properties for hydrolysis of both sucrose and Neosugar were determined. For sucrose and Neosugar respectively, Michaelis constants at 30 mC and pH 6n0 were 7n7p0n5 and 14n1p1n1 mol m −$ (as terminal fructose) and maximum velocities were 6n5p0n1 and 2n7p0n1 mg g −" fr. wt h −" (equivalent to 10n0 and 4n2 nkat g −" as reducing sugar release). Maximal temperatures for activity were 45 and 44 mC, and Arrhenius activation energies were 39n9 and 46n9 kJ mol −" . Preincubations for 1 h at 49 and 48 mC caused 50 % loss of activity in subsequent assays at 30 mC. The pHs for maximal activity for the two substrates were 5n2p0n1 and 5n5p0n1.Using size exclusion chromatography (SEC), an activity catalysing the formation of fructan oligosaccharides and another catalysing sucrose hydrolysis, were not fully resolved, but exhibited distinct profiles of elution indicating M r of 57 and 133 kD respectively. When assayed for the hydrolysis of Neosugar, the SEC eluate exhibited two peaks of activity indicative of M r values of 57 and 133 kD.

Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation

“…The value is similar to previous reports for L. temulentum and other temperate grasses determined for desalted and concentrated enzyme preparations (Cairns and Pollock 1988b;Cairns 1992;Cairns and Ashton 1993;Cairns et al 1997). The extractable soluble activity fully hydrolysed the endogenous sucrose in homogenates of 3-h-illuminated leaves (12 mg g )1 FW) in 4-5 h, as illustrated in Fig.…”

“…When supplemented with exogenous sucrose to the saturating concentration of 200 mM (Cairns et al 1997), reducingsugar release was linear with respect to time over a 2-h period at the maximum velocity (V max ) of 1.9 mg g )1 FW h )1 (Fig. 1b, open circles).…”

“…-Thirdly, we recognise that the inhibitory treatments could have altered vacuolar pH causing a modulation of AI activity. However, we note that the pH optimum for activity for the L. temulentum AI is broad, maintaining ‡80% of maximal activity between pH 4.0 and 6.2 (Cairns et al 1997). Hence, the in vivo activity of the enzyme would be insensitive to vacuolar pH.…”

“…The initial rate of hydrolysis of the endogenous sucrose was 26% lower than V max . This is explicable because the Timings relate to points in a sucrose K m of the AI is 8 mM (Cairns et al 1997). Since the initial substrate concentration in vitro was 17.5 mM, and fell continuously, the activity was initially sucroselimited and the rate was continuously reduced by progressively more severe substrate limitation as the reaction proceeded to completion.…”

“…No desalting step was included and the cleared homogenate was mixed 4+1 with 1.0 M sucrose in the acetate buffer. Release of reducing sugar was determined over a 2-h period using the Somogyi-Nelson procedure to determine V max (Cairns et al 1997). To assess direct effects of the metabolic inhibitors on AI activity, the homogenate was mixed 4+1 with the buffered substrate containing either 25 mM NaVO 3 , 50 mM mannose or 5 mM NaCN (pH 5.3 with acetic acid) or no addition (sucrose-only control).…”