The penetration of α-chymotrypsin and/or pancreatic lipase into dead cells of soybean cotyledons, of yeast and of algae was studied using the enzymic activity as a parameter. In addition a fluorescent antibody technique was applied for the localization of α-chymotrypsin within the soybean cells.
The digestibility of unheated, EDTA-treated substrate was similar to that of the heated substrate. Since EDTA-treatment increases the permeability of the cell wall and cell membrane of the plant cell without denaturing proteins contained in the cells, we concluded that heating affected mainly the barrier formed by the cell wall, thus permitting a better passage of big molecules (enzymes).
Leakage of β-amylase (mol. weight 61,700) from soybean occurred only after treatment with EDTA. This confirmed that EDTA enhances the permeability of the cell wall.
α-Chymotrypsin (mol. weight 24,000) and pancreatic lipase (mol. weight 38,000) penetrated unheated sections, whereas amylopectin (mol. weight 50,000-1,000,000) did not penetrate unheated cotyledons. Apparently the greater dimension of amylopectin compared with α-chymotrypsin and lipase accounts for its lack of entry into unheated soybean cotyledons. However, when we applied the fluorescent antibody technique to localize α-chymotrypsin within the cells from unheated sections, we used antibodies (γ-globulins) having a mol. weight of 150,000. They entered the unheated sections and produced a positive result of the experiment. Consequently the size of the molecules as indicated by the molecular weight, is not the reason why amylopectin did not penetrate the unheated cotyledons. Moreover, this shows that α-chymotrypsin probably opens a way for the entry of globulins. Therefore the entry of proteolytic enzymes is not an inert process. This is conceivable as the enzymes are big molecules with catalytic activity.
Unheated non-EDTA-treated soybean (with plasmodesmata) was slightly penetrated by α-chymotrypsin, whereas unheated non-EDTA-treated yeast and algae (both without plasmodesmata) were not penetrated at all. Furthermore, in soybean material (unheated or heated) α-chymotrypsin enhanced the penetration of lipase; this effect was absent in the case of yeast. The results obtained strongly suggest that plasmodesmata are a way in dead plant cells for penetration of enzymes.