Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two a-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of a-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse-chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra-to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70°C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation.two forms shows differential sensitivity to monensin and Ca2" which led Akazawa and Hara-Nishimura (1) to propose that there may be differential intracellular sorting and transport of the two forms and that this may be based on post-translational glycosylation and the nature of the side chain added. Monensin and Ca2" also differentially affect the secretion of the low pl2 and high pI groups of barley aleurone a-amylase (17, 23) indicating that perhaps there are similarities in the mechanisms of aamylase secretion between the two tissues.The goals of the experiments reported in this paper were to determine whether barley aleurone a-amylase isozymes are posttranslationally modified during intracellular transport and to establish the nature of any such covalent change. Our experiments were prompted by the finding that barley aleurone protoplasts contain forms oflow pI a-amylase which are not secreted (16). We present evidence that these intracellular forms of aamylase are precursors of the secreted forms of the enzyme. We also show that the modification results in a lowering of isozyme pI without significantly affecting the size of the enzyme. We conclude that this post-translational modification is not brought about by glycosylation, although the precise nature of the covalent change remains to be identified. MATERIALS AND METHODSThe cereal aleurone layer is a specialized tissue which synthesizes and secretes hydrolytic enzymes into the starchy endosperm of germinating grain. Although details of the regulation of enzyme synthesis by GA3 are beginning to be understood (12)(13)(14), far less is known about the mechanisms of intracellular transport and secretion of the inducible proteins. a-Amylase is probably synthesized on the endoplasmic reticulum (9, 17), and exocytosis probably occurs v...