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Host-Pathogen Interactions

XVI. PURIFICATION AND CHARACTERIZATION OF A -GLUCOSYL HYDROLASE/TRANSFERASE PRESENT IN THE WALLS OF SOYBEAN CELLS ¹

Department of Chemistry, University of Colorado, Boulder, Colorado 80309

The fact that fungal glucans will stimulate soybeans to accumulate phytoalexins prompted an investigation of soybean cell -1,3-glucanases and -glucosidases, as well as the ability of these enzymes to hydrolyze the fungal glucans. Several -1,3-glucanases and -glucosidases can be solubilized from the walls of suspension-cultured soybean cells by treatment with 1.0 molar sodium acetate buffer. An enzyme, which has been termed -glucosylase I, is the dominant -1,3-glucanase in the cell wall extracts. Utilizing CM-Sephadex chromatography, hydroxylapatite chromatography, and affinity chromatography, -glucosylase I has been purified 71-fold, with 39% recovery, from the mixture of cell wall enzymes. The affinity chromatography column material was prepared by covalently attaching p-aminophenyl-1--D-glucopyranoside, an analog of a -glucosylase I substrate, to Sepharose. -Glucosylase I, purified by this procedure, yields a single band on isoelectric focusing gels (pH 8.9). However, the purified -glucosylase I yields a darkly-staining protein band at an apparent molecular weight of 69,000 and several lightly-staining protein bands in sodium dodecyl sulfate polyacrylamide gels. Additional purification procedures fail to remove these lightly-staining protein bands.

-Glucosylase I will hydrolyze the -glucan substrates, laminarin (3-linked) and lichenan (3- and 4-linked), and therefore, possesses -glucanase activity. Studies of the progressive hydrolysis of laminarin by -glucosylase I demonstrate that the enzyme hydrolyzes polysaccharide substrates in an exo manner. -Glucosylase I will also hydrolyze a variety of low molecular weight -glucosides including various -linked diglucosides. Thus, -glucosylase I also possesses -glucosidase activity.

Several lines of evidence are presented that the -glucanase and the -glucosidase activities exhibited by purified -glucosylase I preparations are catalyzed by the same enzyme. This evidence includes inhibition studies which indicate that the -glucanase and the -glucosidase activities of -glucosylase I are catalyzed at the same active site. -Glucosylase I will also catalyze glucosyl transfer. This catalytic activity is responsible for the observed ability of the enzyme to synthesize di- and trisaccharides from laminarin. The disaccharides formed by -glucosylase I-catalyzed transglucosylation are the -anomers of the 6-, 4-, 3-, and 2-linked diglucosides in the relative proportions of 10:1:1:1. The ability of -glucosylase I to catalyze glucosyl transfer indicates that -glucosylase I is biochemically more similar to previously studied -glucosidases than to -glucanases. This conclusion is supported by the observation that -glucosylase I is strongly inhibited by 1,5-D-gluconolactone, an inhibitor of -glucosidases but not of -glucanases.

³ To whom correspondence should be addressed.

¹ Supported by the Department of Energy, Contract EY-76-S-02-1426.

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