Purification and characterization of a serine protease from senescent wheat leaves

ROBERTS, IRMA N; FERNANDEZ MURRAY, PEDRO; CAPUTO, CARLA; PASSERÓN, MARÍA SUSANA; BARNEIX, ATILIO J

PHYSIOLOGIA PLANTARUM

Año: 2003 vol. 118 p. 483 - 490

0031-9317

A senescence-specific protease accounting for almost 70%of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent K m and V max for the peptide were 1.18 mM and 2.27 mmol pNA mg/h respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60 C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity .Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65 –75%identity)with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process.