β-D-glucan-hydrolase activities in pure cell-wall-enriched fractions from Valerianella olitoria cells

An effective method for the preparation of purified cell walls from mesophyll cells of Valerianella olitoria has been developed. Cells were isolated by a mechanical procedure only and crude cell walls were prepared from cell homogenates. Crude wall suspensions were fractionated in a discontinuous sucrose gradient and the wall fragments recovered were examined by scanning electron microscopy. An evaluation of the degree of purity and physiological integrity of the wall fragments showed that the material found at the 50-60% (w/w) interface consisted mostly of wall particles of high purity. Some characteristics of the purified walls are reported, especially the following enzyme activities: β-D-glucosidase (EC 3.2.1.21) and the β-D-glucanases, 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), 1,4-β-glucan cellobiohydrolase (EC 3.2.1.91), 1,3-β-glucan glucanohydrolase (EC 3.2.1.39), 1,3-β-glucan glucohydrolase (EC 3.2.1.58). The results provided evidence for the microlocalization of some hydrolases and indicated that enzymes extracted only with a high-salt-concentration buffer were confined to walls whereas the 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-solubilized enzymes could have multiple sites, e.g. walls and membranes of the endoplasmic reticulum.