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

Abstract

A re-examination of the kinetic properties of UDP-glucose: (1-.3)-i% 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 j-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V. of the enzyme, and by lowering the apparent K,. for UDPglucose 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 fglucoside; with saturating Ca2', Mg2' only slightly stimulates further production of glucan. However, the presence of Mg2' during synthesis, or NaBH. 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 Ca24, #-glucoside-activated cotton fiber enzyme, with or without Mg2', is very similar in size to callose isolated from cotton fibers, but is a linear (1-.3)-#-glucan lacking the small amount of branches at C0-6 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 centrifuption of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.Essentially all higher plants contain a UDP-glucose: (1-+3)-f3-glucan (callose) synthase. This enzyme is largely found on the plasma membrane, and in most cases is latent and only becomes activated by perturbed conditions which lead to some loss of membrane permeability (5, 16). Some years ago, Ray (26) named this enzyme Glucan Synthetase II, and assayed it at high concentrations of UDP-Glc in the absence of divalent cations, although others have reported that activity can be enhanced by Mg2' (6),

Cotton fiber annexins: a potential role in the regulation of callose synthase

“…However, we cannot rule out the possibility that purified callose synthase may still possess associated phospholipid (Wasserman and McCarthy, 1986) which could facilitate the interaction. The evidence presented here indicating an interaction between callose synthase and annexin support the conclusion that the cationdependent changes in size of callose synthase observed previously (Hayashi et a/., 1987), are most likely due to association of the enzyme with p34. Other studies of ours (Herth and Delmer, manuscript in preparation) indicate that another membrane-localized non-catalytic subunit of 65 kDa also associates with the enzyme under these conditions, but its function remains unknown.…”

“…The enzyme requires both Ca2+ and a p-glucoside for activity, and is normally latent 763 (Delmer, 1987;Kauss, 1990). The detergent-solubilized enzyme has been shown to assume a larger size under the influence of divalent cations, a property which was suggested to be due either to aggregation or a cationdependent, reversible association with additional polypeptides (Hayashi et al, 1987). Activation of the enzyme during wounding is presumed to result from transient rises in the level of intracellular Ca2+ (Kauss, 1990) and, perhaps also, in the level of a natural p-glucoside activator such as p-furfuryl-p-glucoside (Ohana et al, 1991(Ohana et al, ,1992.…”

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…There are at least nine clades of nucleotide sugar pyrophosphorylases ( Figure 1A), which have different activities and physiological functions (Kotake et al, 2010). UDP-Glc pyrophosphorylases generate UDP-Glc, the starting substrate of UDP-sugar metabolism, which is used for the synthesis of cellulose and b-1,3-glucan and for the b-1,4-glucan backbone of xyloglucan (Hayashi et al, 1987;Gordon and Maclachlan, 1989;Cocuron et al, 2007;Park et al, 2010). A plant-specific enzyme, UDP-sugar pyrophosphorylase (USP), converts various monosaccharide 1-Ps to their respective UDP-sugars in the salvage pathway and is required for pollen development (Kotake et al, 2004(Kotake et al, , 2007Schnurr et al, 2006).…”

Separation and Partial Purification of 1,3-β-Glucan and 1,4-β-Glucan Synthases from Saprolegnia

“…In the present paper, we report on the partial purification of 1,3-f3-glucan and 1,4-13-glucan synthases. Enzymes pelleted by an entrapment procedure (12) with the reaction product were solubilized and then purified by density gradient centrifugation.…”