Characterization of oat endoproteinases that hydrolyze oat globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characteriz e these endoproteinases. A qualitative PAGE method that used oat globulins as gel-incorporated substrates was used to determine which enzymes hydrolyz ed the globulins. The proteolytic hydrolysis products were studied by hydro lyzing the globulins in vitro with the endoproteinases and analyzing the pr oducts by SDS-PAGE. Class-specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine-class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major ac tivity only appeared on the sixth day of germination. Extracts from four-da y germinated oats contained cysteine proteinases that hydrolyzed the globul ins in vitro to form a polypeptide of intermediate size (MW approximate to 34,500). Cysteine proteases from an eight-day germinated sample totally hyd rolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurr ed quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value wh ere the globulins can be degraded.