THE RELEASE OF BOUND BETA-AMYLASE BY MACROMOLECULES

The aim of this study was to clarify the roles of endogenous proteases and higher molecular weight thiols in the release of bound beta-amyla se, which occurs during barley germination. In resting barley grains ( Hordeum vulgare L. cv. Torrent) five major beta-amylase monomers were found in the thiol reduced, salt soluble extract. Only the two smalles t monomers were present in large amounts in the bound form of the enzy me that had been released with 5-mercaptoethanol. In vitro, bound beta -amylase was solubilised by ''releasing factors'' extracted from the e ndosperm of decorticated grains germinated for three days. The releasi ng factors were in the higher molecular weight fraction (>5 kDa) and t heir formation was induced in degermed grains by gibberellic acid, Whe n an endosperm extract, containing only higher molecular material (>5 kDa) prepared from three day germinated grain. was incubated with a pr eparation of bound beta-amylase, about 75% of the release of the enzym e could be prevented by a mixture of proteolytic inhibitors, The domin ant class of proteases in malt were the sulphydryl proteases. These we re not fully active when extracted, but could be activated approximate ly six fold by the addition of S-mercaptoethanol. Heating the extract to a limited extent destroyed all the proteolytic activity. but 10% of the bound beta-amylase could still be released by the extract.