Release and Activity of Bound beta-Amylase in a Germinating Barley Grain.

In resting grains of Triumph barley (Hordeum vulgare L. cv Triumph) about 40% of the beta-amylase could be extracted with a saline solution, the remaining 60% being in a bound form. During seedling growth (20 degrees C), the bound form was released mainly between days 1 and 3. When a preparation containing bound beta-amylase was incubated with an extract made of endosperms separated from germinating grains, release of bound beta-amylase took place and could be studied in vitro. The release was almost completely prevented by leupeptin and antipain, specific inhibitors of a group of SH-proteinases, but it was not inhibited by pepstatin A or EDTA, which inhibit some other barley proteinases. It is thus very likely that in a whole grain, at least the bulk of the bound beta-amylase is released by the proteolytic action of one or several SH-proteinases. When the bound beta-amylase was released by papain, its molecular weight was about 5000 daltons smaller than that of beta-amylase released by dithiothreitol. This indicates that the release is due to removal of a sequence of beta-amylase itself. A similar decrease in size took place during seedling growth. Bound beta-amylase showed some activity against native starch and it hydrolyzed maltotetraose at a rate that was about 70% of the rate the same amount of bound beta-amylase gave after release. Bound beta-amylase is thus not inactive and it is likely that the slower rate of hydrolysis is due to steric hindrances which prevent substrates from reaching the active site.