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

• Published in: The international journal of biochemistry & cell biology Vol. 32; no. 11; pp. 1195 - 1204
• Main Authors: Iulek, Jorge, Franco, Octávio Luiz, Silva, Márcio, Slivinski, Christiane Trevisan, Bloch, Carlos, Rigden, Daniel John, Grossi de Sá, Maria Fátima
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.11.2000
• Subjects: Acanthoscelides; alpha-Amylases - antagonists & inhibitors; Animals; Bifunctional α-amylase/trypsin inhibitor; Chromatography, Ion Exchange; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - isolation & purification; Enzyme Inhibitors - pharmacology; Humans; Molecular Sequence Data; Molecular Weight; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; Rye; Secale - chemistry; Serine Proteinase Inhibitors - pharmacology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin Inhibitors - pharmacology; Zabrotes; α-Amylase inhibitor; Bifunctional α-amylase/trypsin inhibitor; Zabrotes; Acanthoscelides; α-Amylase inhibitor; Rye
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/S1357-2725(00)00053-4

Plant α-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the α-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13 756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional α-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus α-amylases was observed. The inhibitor is more effective against insect α-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional α-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.