Partial purification of endo- and exo-?-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis

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Abstract
The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 μg/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities. Molecular-sieve chromatography of the cell-wall protein resolved endo-β-D-glucanase and exo-β-1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo-β-D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan. Cell-wall β-D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze β-D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of β-D-glucan in vitro. During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall β-D-glucan, the primary substrate for autolytic reactions.