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)(2)SO4 fractionati on, gel filtration, and both ion-exchange and hydrophobic chromatograp hy. Constancy of the specific activity of chromatographic fractions an d their SDS-electrophoretic pattern indicates the homogeneity of the f inal enzyme preparation. However, electrophoresis in nondenaturing con ditions revealed three protein bands of similar intensity, each showin g proteolytic activity. The N-terminal sequences of all three electrop horetic components are identical. They are also identical to a segment of the amino acid sequence deduced from one of several cDNA clones de rived from closely related, but non-identical mRNAs that accumulate in the aleurone layer of gibberellic acid-treated wheat [1]. It is very likely that the three electrophoretic components found are isoenzymes encoded by cDNA clones described by these authors. (C) 1997 Elsevier S cience Ltd. All rights reserved.