PURIFICATION, CHARACTERIZATION AND ACTIVATION BY ANIONS OF AN ASPARTIC PROTEINASE ISOLATED FROM BRAN OF SOFT WHEAT

An aspartic proteinase, contained in wheat bran, was purified to homogeneity by ion-exchange, gel permeation, affinity chromatography and electro-endosmotic preparative electrophoresis. The optimal pH for the activity of the purified enzyme was 3.3, as measured by the hydrolysis of a specific chromophoric substrate, and the K-m value was 0.375 mM. The apparent molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 66.5 kDa. The enzyme activity was completely inhibited by pepstatin, whereas other proteinase inhibitors were ineffective. The enzyme hydrolyzed at pH 3.3 endogenous globulin, releasing fragments with molecular weights over 20 kDa. The purified proteinase was activated by anions, particularly sulfate, and this effect was instantaneous, operative when the reaction was initiated, and insensitive to changes in the ionic strength of the reaction medium. A comparison with other aspartic proteinases of animal origin revealed that the enzyme modulation by anions was a characteristic of the plant enzyme alone. Finally, circular dichroism analyses showed a conformational change in the plant enzyme in the presence of sulfate. These data suggest a new control mechanism for modulating proteolytic activity in seeds, and the participation of aspartic proteinase in initiating the degradation of globulin stored in the aleurone layer.