The CM2 and CM3 types of α-amylase inhibitor are associated with Triticum aestivum seed chromatin

Treating the crude chromatin fraction of wheat seed (Triticum aestivum L.) with alkali and methanol solubilizes several small phosphorylated peptides and a protein fraction, which is almost pure in native and SDS-PAGE analysis. The protein material is composed of two polypeptides, which can be easily separated in PAGE in the presence of 8 M urea, 1 mM 2-mercaptoethanol, 5% (v/v) acetic acid (pH 4.5) and 0.1 (%) v/v Triton X-100. N-terminal sequence analysis revealed that the two polypeptides have sequences that are identical to that of the secreted form of the so-called wheat seed CM2 and CM3 proteins, respectively, belonging to the α-amylase inhibitor superfamily. Their inhibitory activity has been determined in vitro using human salivary amylase and bovine pancreatic trypsin. Immunoblotting experiments, carried out with an antiserum raised in rabbit and which recognizes both CM2 and CM3 proteins, indicate that the protein is mainly expressed in seeds rather than in roots and coleoptiles. In addition, western blotting analysis of protein extracted from highly purified wheat seed chromatin preparation revealed immunoreactivity at the level of the CM2/CM3 protein bands. The possible function of these proteins at the chromatin level has been investigated using gel shift assay and the effect on RNA transcription in vitro. The results indicate that the CM2/CM3 protein binds DNA and inhibits in vitro RNA transcription, suggesting that the protein has a role in the control of the chromatin activity.