Purification and characterization of a serine protease from senescent wheat leaves.

ROBERTS, IRMA; CAPUTO, CARLA; FERNANDEZ MURRAY, PEDRO; PASSERON, SUSANA; BARNEIX, ATILIO; CARLA PAULA CAPUTO SUAREZ

VILLA CARLOS PAZ CORDOBA

Congreso; XXXVIII Reunión Anual de SAIB; 2001

A protease representing almost 50% of total proteolytic activity in protein extracts of senescent wheat leaves, not detectable in fractionated extracts from non-senescent tissues, was isolated from dark-induced senescent leaves. Plants were grown in a culture chamber with 10 mM NO3- nutrient solution and a photoperiod of 16 h. Leaves from 15-days old plants were detached and incubated in continuous darkness for 3 days. They were used for preparation of crude extracts. Purification was performed by ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose, Q-Sepharose and gel filtration chromatography. The enzyme was followed by its ability to hydrolyse azocasein at pH 7.5. When other substrates were used, the protease resulted active against the peptide N-succinyl-AAPFp-NA, a substrate for chymotrypsin. Also, the enzyme was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. The azocaseinolytic activity was strongly inhibited by PMSF but not by E-64, suggesting that the enzyme might belong to the serine proteases class. SDS-PAGE analysis of the purified preparation showed one protein band of an apparent molecular mass of approximately 65 kDa.