Studies on a-amylase inhibitors from seeds of sorghum bicolor

Abstract

Six inhibitors (Slal, SIa2, SIa3, SIa4, SIa5 and SIa6) of a-amylase from mammalian, insect, bacterial and fungal sources were purified from seeds of Sorghum bicolor (L) Moench by saline extraction, precipitation with ammonium sulphate, affinity chromatography on Red Sepharose, preparative and analytical reverse phase HPLC on Vydac C(_18) columns. The complete primary structures of five of these inhibitors (Slal-5) were determined by automatic degradation of the intact, reduced and S-alkylated proteins and by manual DABITC/PITC microsequencing of peptides obtained from enzyme digests. The first three inhibitors consist of 47 (Slal) and 48 (SIa2, SIa3) amino acids with respective molecular weights of 5,396, 5,310. and 5384. These basic proteins (pi predictions above 8) were found to be highly homologous between themselves and with the recently isolated γ-hordothionin. γ(_1)-and γ(_2)-purothionins (Colilla et al., 1990; Mendez et al., 1990) and are, therefore, considered to be thionin-like inhibitors. Four disulphide bonds were identified and their positions determined in the sequence of Slal. It has been reported that the a-amylase inhibitory activity of thionins is due to competition for calcium ions which is the most important co-factor for this enzyme activity (Matsuura et al., 1984; Buisson et al., 1987). Calcium binding motifs have been located in the sequences of Slal, Sla2 and Sla3 and their structural significance has been investigated by molecular modelling.SIa4 and SIa5 which consist of 118 (MW 12,485) and 116 (MW 12,761) amino acids respectively are also basic polypeptides (pI predictions above 8). These proteins were found to be 35% homologous between themselves and showed significant homology (range 21-42%) with the members of the cereal superfamily. Hydrophobicity plots and secondary structure prediction results also revealed common features between these proteins and those of the cereal superfamily. Only a preliminary N-terminal sequence was obtained for Sla6 which was found to inhibit human salivary a-amylase and locust gut a-amylase.