RELEASE AND ACTIVATION OF BARLEY BETA-AMYLASE

The aim of this study was to determine the role of low molecular weigh t thiols both in the release and activation of beta-amylase during gra in germination. In quiescent barley grains (Hordeum vulgare L. cv. Tor rent) about 55% of the beta-amylase was extracted with buffer. the rem aining 45% was in the bound form, During micromalting the bound form w as progressively solubilised between germination days 1 and 4. When fr ee beta-amylase, extracted from ungerminated grains, was incubated wit h dithiothreitol the enzymic activity increased by 15%-20%. This activ ation did not occur when free beta-amylase, from grain germinated for 3 days or more, was incubated with DTT. The release of bound beta-amyl ase with thiols was pH dependant, occurring most rapidly at and above pH 8.0. At the onset of germination the embryo released soluble thiol (approximately 5 nmol per embryo) into the endosperm. Degermed grains were dosed with reduced glutathione and incubated for 72 h. The additi on of 60 nmol glutathione caused the release of about 80% of the bound beta-amylase. When less glutathione was used, 5 nmol (an amount simil ar to that released by the embryo in vivo) no significant release of t he bound enzyme was detected. When degermed grains were dosed with oxi dised glutathione (60 nmol), no bound beta-amylase was released. Howev er, addition of the disulphide bis-hydroxyethyldisulphide (60 nmol) di d cause the release of about 90% of the bound enzyme. The aleurone lay er reduced the bis-hydroxyethyldisulphide to a thiol, presumably 2-mer captoethanol. Oxidised glutathione and cystine were not significantly reduced to thiols by isolated aleurone layers. The aleurone layer did cause the disappearance of cysteine from solution. When preparations o f bound beta-amylase were incubated with extracts from the endosperms of grains germinated for three days, the bound enzyme was released. Th is release was due to the high molecular weight material (>5 kDa) in t he extract and not to low molecular weight thiols. It seems unlikely t hat simple thiols. such as glutathione, are solely responsible for the release of bound beta-amylase.