The amino acid sequences of two 13-kDa alpha-amylase inhibitors from the seeds of Sorghum bicolor (L.) Moench.

Abstract

Two inhibitors (SI alpha 4 and SI alpha 5) of the alpha-amylases from insect and mammalian sources were purified from seeds of Sorghum bicolor by saline extraction, precipitation with ammonium sulphate, affinity chromatography on Red Sepharose, and preparative and analytical reverse-phase HPLC on columns of Vydac C18. The complete primary structures of these two inhibitors were determined by automated degradation of the intact, reduced and S-alkylated proteins and by manual 4-N,N-dimethylaminoazobenzene-4-isothiocyanate/phenyl isothiocyanate microsequencing of peptides derived from them following enzyme digests. The amino acid sequences were as follows: SI alpha 4: TVDVTACAPGLAIPAPPLPTCRTFARPRTCGLGGPYGPVDPSPVLKQ- RCCRELAAVPSRCRCAALGFMMDGVDAPLQDFRGCTREMQRIYAVSRLTRAAECNLPTIPGGGCHLSNS PR; and SI alpha 5: ANWCEPGLVIPLNPLPSCRTYMVRRACGVSIGPVVPLPVLKERCCSELEKLV- PYCRCGALRTALDSMMTGYEMRPTCSWGGLLTFAPTIVCYRECNLRTLHGRPFCYALGAEGTTT. Comparisons of these sequences with one another and with those of other proteins in the US National Biomedical Research Foundation Databank indicated that the two Sorghum proteins had significant similarities (21%-42% identity) with the members of the cereal superfamily of enzyme inhibitors.