Post-translational N- and C-terminal processing in the development of wheat leaf β-amylase polymorphism from an hitherto undetected primary gene product

The β-amylase (EC 3.2.1.2) of wheat (Triticum aestivum L.) leaves comprises at least five distinguishable isoforms (βI–βV) apparently corresponding to a β-amylase-encoding cDNA obtained from leaf mRNA (cDNA1). This β-amylase has now been purified by immunoaffinity chromatography on antibodies directed against the heterologous expression product of cDNA1. Upon resolution into component forms by anion exchange chromatography, a new β-amylase form larger than any of βI–βV was discovered (βN). The electrophoretic patterns and amino acid sequences of peptides yielded by tryptophan-specific digestion confirmed that the leaf β-amylase isoforms all correspond to cDNA1. C-terminal peptides of the isoforms showed the same size progression as did the intact proteins (βN > βV > βIV > βIII > βII > βI). According to mass spectrometry, βV, βIII/βII and βI represent truncations of 8, 12 and not more than 14 amino acids, respectively, of the C-terminus of βN. All of the β-amylase forms begin N-terminally with the (acetylated) Ala2 of the amino acid sequence predicted by cDNA1, although some of each of βI–βIII also commence with Met5. A leaf β-amylase-encoding gene corresponding to cDNA1 is thus translated under removal of the initiator methionine and acetylation of the newly formed N-terminus to produce βN. βI–βV are then formed from βN by the removal of up to 14 amino acids from the C-terminus, concomitant to the removal of three further N-terminal amino acids during the formation of some of βI–βIII.