Abstract
A re-examination of the kinetic properties of UDP-glucose: (1-->3)-beta-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 Ca(2+) and millimolar concentrations of beta-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V(max) of the enzyme, and by lowering the apparent K(m) for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg(2+) markedly enhances the affinity of the mung bean enzyme for Ca(2+) but not for beta-glucoside; with saturating Ca(2+), Mg(2+) only slightly stimulates further production of glucan. However, the presence of Mg(2+) during synthesis, or NaBH(4) 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 Ca(2+), beta-glucoside-activated cotton fiber enzyme, with or without Mg(2+), is very similar in size to callose isolated from cotton fibers, but is a linear (1-->3)-beta-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 Mg(2+)in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg(2+) 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.