Chain Initiation in Glucan Synthesis in Mung Beans

Abstract

Feingold et al. (1958) and Flowers et al. (1968) have shown that a soluble enzyme fraction from mung beans (Phaseolus aureus) catalyses the formation of callose, a structural p-(I +3)-glucan, from UDP-glucose. Glucans synthesized under these conditions do not contain 8-(1+4)-linkages. Since this enzyme is soluble any substance needed to initiate a polysaccharide chain must be present in solution in the enzyme extract, so that an examination of the mechanism of priming should be possible. The solubilized enzyme was prepared from mung-bean shoots aged 4+ days by using the method described by Flowers et al. (1968). After incubation of the enzyme for lOmin in 2m~-MgC1, in 80mM-Tris-HCI buffer, pH7.5, with 1 ~ ~ U D P [ ' ~ C ] g h ~ c o s e about 80% of the radioactivity was incorporated into a compound that had chromatographic properties similar to those of laminaribiose but could be distinguished from it. Hydrolysis of the unknown compound, designated biose, by HCI or by treatment with a glucanase gave glucose as the only radioactive product, as did oxidation by lead tetraacetate followed by hydrolysis. The biose was not reduced by NaBH4. These lines of evidence suggested that only one sugar unit was radioactive, the other not being formed from the sugar nucleotide, and also that the biose was a non-reducing compound. Further identification depended on the production of microgram quantities of the biose. This was not easy because the non-radioactive moiety, presumably supplied by the enzyme extract, was limiting the reaction. In certain experiments variable amounts of a triose were formed, and this was also isolated for analysis. G.1.c. of the trimethylsilyl derivative of the biose gave a single peak, indicating that the compound was non-reducing. Calculation of the peak height after the injection of a known amount of radioactivity gave an estimate of the specific radioactivity and confirmed that only one sugar moiety contained 14C. Mass spectrometry indicated that the biose was not a reducing sugar (see Kochetkov et al., 1968) but that its molecular weight was that of a dihexose. An acid hydrolysate of the biose and of the triose was trimethylsilylated and the components of each were separated by g.1.c. Each preparation appeared to give the same three peaks, which were characterized more fully in the sample from the triose. Two of the three peaks did not separate from the aand &peaks of trimethylsilylglucose and the third did not separate from the derivative of myoinositol. In the biose the ratio of the peak heights was that expected for 1 molecule of glucose and 1 of inositol, whereas in the triose there was twice as much glucose as inositol. I t therefore appears that the biose was glucosylinositol and the triose diglucosylinositol. Addition of [3H]myoinositol with UDP-[*4C]glucose to the enzyme digest resulted in the incorporation of small amounts of 3H into the biose. The amount of inositol incorporated was only a very small proportion of the aglycone of the biose made, but the amount of added inositol did increase at higher concentrations of UDP-glucose and inositol, indicating that free inositol was not the immediate substrate for synthesizing the biose but supporting the characterization of the biose as glucosylinositol. When the enzyme was isolated from older plants it was possible to show that, after prolonged incubation under the conditions described above, the amount of biose and triose decreased and the amount of higher oligosaccharides increased (Fig. 1). When the [14C]triose was incubated with non-radioactive UDP-glucose, radioactive higher oligosaccharides were formed of which a small proportion was insoluble in water. Clearly, therefore, glucosylinositol can act as a primer in glucan synthesis. Preparations from some plants did not appear to convert the biose into a polymer, a property that appeared to be connected with the age of the plant. Seedlings aged 44-5 days did not use the biose as primer, whereas older or younger seedlings always did. Older seedlings also made the biose, but this did not accumulate to such an extent. Under conditions when the biose was not converted into higher oligosaccharides there was no formation of glucan, and it appears that, in this system, all the polysaccharide is formed de novo. Since callose