Detection of the isoenzymes of wheat grain proteinase A

A new method for the purification of wheat cysteine proteinase A which plays a key role in the mobilization of seed storage proteins during germination has been developed. It consists of (NH4)2SO4 fractionation, gel filtration, and both ion-exchange and hydrophobic chromatography. Constancy of the specific activity of chromatographic fractions and their SDS-electrophoretic pattern indicates the homogeneity of the final enzyme preparation. However, electrophoresis in nondenaturing conditions revealed three protein bands of similar intensity, each showing proteolytic activity. The N-terminal sequences of all three electrophoretic components are identical. They are also identical to a segment of the amino acid sequence deduced from one of several cDNA clones derived from closely related, but non-identical mRNAs that accumulate in the aleurone layer of gibberellic acid-treated wheat. It is very likely that the three electrophoretic components found are isoenzymes encoded by cDNA clones described by these authors.