Endopeptidases during the development and senescence of Lolium temulentum leaves

The endopeptidase activity of Lolium temulentum leaf tissue was measured using azocasein as a substrate. The enzyme increased with leaf age, and also during senescence of excised leaf tissue. There were at least two distinct endopeptidase activities, characterized by different pH optima. The predominant form in leaves of intact plants was maximally active at pH 5. In detached leaves during the later stages of senescence this activity was replaced by an enzyme with an optimum at pH 8. An antibody raised against the non-glycosylated cysteine endopeptidase papain cross-reacted with polypeptides in protein preparations of L. temulentum leaf tissue. The correlation between enzyme activity and the pattern of immunoreactive polypeptides suggested that the polyclonal antibody was able to recognize the Lolium homologues of papain. The switch from pH 5 to pH 8 enzyme in detached leaves was associated with an evident decrease in the M(r) values of papain-like antigens detected on western immunoblots, from ca 60 000 to 30 000. It is possible that the alkaline activity is derived from the acid form, perhaps by limited autolysis in protein-depleted tissue at an advanced stage of senescence. On the other hand, the response of protease activation to treating leaf tissue with inhibitors of protein biosynthesis is more consistent with de novo appearance of a different form of the enzyme in late senescence.