Purification and characterization of an endoprotease from alfalfa senescent leaves

We purified and characterized an endoprotease, whose activity increased during growth and senescence of alfalfa (Medicago sativa L.) leaf. The purification and characterization of proteolytic enzymes is a helpful step to understand their function and involvement in the process of protein degradation during foliar senescence. One of the two proteases, revealed after affinity chromatography on haemoglobin-agarose, was characterized. Physical and biochemical properties of the purified protease were identified and the effects of various inhibitors on enzyme activity were tested. The protease activity, with a pH optimum at 5.0, was affected by metallo-protease inhibitors and enhanced by organomercurial compounds. The purified protease was tested on ribulose-1,5-bisphosphate carboxylase/oxygenase, and the electrophoretic analysis of the reaction mixture showed that the large subunit decreases during the incubation, indicating that the purified protease is capable of degrading the large subunit in vitro. A possible involvement of such protease in the process of protein degradation during leaf senescence can be considered. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.