Complete primary structures of two subunits of purothionin A, a lethal protein for brewer's yeast from wheat flour.

Abstract

Purothionin (a lethal protein for brewer's yeast) obtained from wheat flour (Triticurm vulgare, Manitoba No. 3) was separated into two active fractions, A and B, by CM-cellulose column chromatography. The main component, purothionin A, was purified to a homogeneous state as shown by polyacrylamide gel disk electrophoresis and isoelectric focusing. The molecular weight of purothionin A was estimated by the sedimentation equilibrium method to be 11,300 in its native state, 5,500 in 6 M guanidine HCl and after its modification with succinic anhydride. Chemical studies including the determination of N- and C-terminal amino acid residues and amino acid sequence analyses using a combination of automatic Edman degradation and tryptic digestion of purothionin A revealed its dimeric structure composed of nonidentical and closely similar polypeptide chains. Two distinct polypeptides were isolated by DE-52 column chromatography after succinylation. Based on the amino acid composition of each polypeptide, it was possible to align all the tryptic peptides of purothionin A without any ambiguities, establishing the complete primary structures of the purothionin A subunits. The two polypeptides in purothionin A have very similar structures, differences occurring at the five positions enclosed by boxes.