UDP-Glucose: (1→3)-β-Glucan Synthases from Mung Bean and Cotton

Abstract

A re-examination of the kinetic properties of UDP-glucose: (13)- -glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca2+ and millimolar concentrations of -glucosides or other polyols is highest at low (1 millimolar to 0.2 to 0.3 millimolar. Mg2+ markedly enhances the affinity of the mung bean enzyme for Ca2+ but not for -glucoside; with saturating Ca2+, Mg2+ only slightly stimulates further production of glucan. However, the presence of Mg2+ during synthesis, or NaBH4 treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca2+, -glucoside-activated cotton fiber enzyme, with or without Mg2+, is very similar in size to callose isolated from cotton fibers, but is a linear (13)- -glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg2+ in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg2+ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme. Images: Fig. 5: