PURIFICATION AND CHARACTERIZATION OF A CYSTEINE PROTEINASE INVOLVED IN GLOBULIN HYDROLYZATION IN GERMINATED VICIA-FABA L

A cysteine proteinase (EC 3.4.22.-), present in high amounts in the co tyledons of germinated seeds of Vicia faba L. 14 d after imbibition, w as purified to homogeneity based on one-dimensional sodium dodecyl sul phate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensiona l PAGE (isoelectric focusing followed by SDS-PAGE). The proteinase has an apparent molecular mass of 31 kDa, an isoelectric point of 4.5, ha s optimal activity at pH 5.0-5.5 and 30-40 degrees C. Classification a s a cysteine proteinase was determined via the effects of numerous pro teinase inhibitors on the activity of the enzyme against the synthetic substrate azocasein. Using native substrates in in vitro digestion an alyses, the proteinase was found to be most active against the higher molecular mass subunits of vicilin and the acidic subunits of legumin, those subunits that are noticeably modified during the early to mid-s tages of storage protein mobilization in vivo. The proteinase shows a high degree of specificity for native and non-native globulin substrat es, but is not capable of modifying either zein (a prolamin) or bovine serum albumin. The peptide complement following in vitro digestion of native substrates resembles that seen during in vivo mobilization of the storage proteins, suggesting that this proteinase is invoked in th e initial modifications of the storage proteins in vivo.