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Metabolism and Enzymology

UDP-Glucose: (13)--Glucan Synthases from Mung Bean and Cotton

Differential Effects of Ca²⁺ and Mg²⁺ on Enzyme Properties and on Macromolecular Structure of the Glucan Product

ARCO Plant Cell Research Institute, Dublin, California 94568-2685, Department of Botany, University of Texas, Austin, Texas 78713-7640

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 (<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. 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.

² Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K.

³ Present address: Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

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