Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha -amylase inhibitors show great potential as tools to engineer r esistance of crop plants against pests. Their possible use is, however, com plicated by the observed variations in specificity of enzyme inhibition, ev en within closely related families of inhibitors. Better understanding of t his specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha -amylase inhibitor famil y of cereal endosperm has been purified from rye using two ionic exchange c hromatography steps. It has been characterised by mass spectrometry inhibit ion assays and N-terminal protein sequencing. The results show that the inh ibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisatio n and is probably glycosylated. The inhibitor has high homology with the bi functional alpha -amylase/trypsin inhibitors from barley and wheat, but muc h poorer homology with other known inhibitors from rye. Despite the homolog y with bifunctional inhibitors, this inhibitor does not show activity again st mammalian or insect trypsin, although activity against porcine pancreati c, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha -amylases was observed. The inhibitor is more effective against insect alp ha -amylases than against mammalian enzymes. It is concluded that rye conta ins a homologue of the bifunctional alpha -amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised. (C) 2000 Els evier Science Ltd. All rights reserved.