Use of Pectic Enzymes in a Study of the Nature of Intercellular Cement of Tobacco Leaf Cells.

Pectic substances have generally been thought to be the principal binding agents between plant cells (12). These pectins are considered to be largely pectic acid because the middle lamella stains with ruthenium red (8). Ginzberg (7) considers that protein and metals are also important binding agents between the cells of the root tip of Alaska pea seedlings. There is, however, a need for further examination of the middle lamella. Setterfield and Bayley (12) in a recent review state, "it has been widely accepted that the middle lamella is principally composed of pectates, but the evidence needs reexamination as it rests mainly on nonspecific extraction and staining procedures." Recently, one of us (14) has devised a technique to separate and isolate living mesophyll cells from tobacco leaves with a commercial pectinase preparation. This preparation, derived from a culture filtrate of Rhizopus tritici, contains a number of enzymes in addition to those which hydrolyze pectins. We have further purified an enzyme from this material with enhanced activity in separating cells. From a study of the substrate specificity of these enzymes we have concluded that the binding of tobacco leaf mesophyll cells is indeed largely a function of pectic acid. Calcium ions appear also to be associated with the intercellular cement.