Characterization of solubilized forms of bound beta-amylase released by various agents

In a previous study the patterns of release of bound beta-anzylase caused b y various agents were investigated. In the present study the released beta- amylase isoforms were purified and characterized The agents used to release bound beta-amylase at different temperatures were papain, thiols, starchy endosperm extract and several substances with amphipathic characteristics. The isoforms released were compared to those present in soluble beta-amylas e from 8-day malt. They were characterized by sodium dodecyl sulphate-polya crylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), nativ e polyacrylamide gel electrophoresis (PAGE), western blotting and attempts were made to use matrix assisted Inter desorption ionization (MALDI) mass s pectrometry. beta-Amylase released by papain was similar in molecular weight (57 kDa by SDS-PAGE) to soluble beta-amylase from 8-day malt (56 kDa). The isoforms re leased by thiol reagents were larger (57-58 and 60-62 kDa) and were similar to the soluble beta-amylase extracted from barley (56-57 and 60-61 kDa) Ag ents with amphipathic characteristics (bovine serum albumin and the deterge nts CHAPS and Triton X-100) released higher molecular weight material (62 k Da). Extracts front starchy endosperm containing proteolytic activity relea sed a beta-amylase isoform, intermediate in size (58 kDa) between that rele ased by papain and those released by thiol-containing and amphipathic agent s. Heat-treated starchy endosperm extract (lacking protease activity) cause d limited (15%) release of beta-amylase isoforms (58 and 61 kDa). These res embled the isoforms released by thiol and hydrophobic agents. The beta-amyl ase isoforms exhibited heterogeneity when analyzed by IEF, native PAGE and MALDI. The results confirm that a complex combination of mechanisms is invo lved in the release of bound beta-amylase during the germination of barley.