Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves

Abstract

Senescence-related proteases play important roles in leaf senescence by regulating protein degradation and nutrient recycling. A 98.9kDa senescence-related protease EP3 in wheat leaves was purified by ammonium sulfate precipitation, Q-Sepharose fast flow anion exchange chromatography and gel slicing after gel electrophoresis. Due to its relatively high thermal stability, its protease activity did not decrease after incubation at 40°C for 1-h. EP3 protease was suggested to be a metal-dependent serine protease, because its activity was inhibited by serine protease inhibitors PMSF and AEBSF and metal related protease inhibitor EGTA. It was identified as a subtilisin-like serine protease of the S8A family based on data from both mass spectrometry and the cloned cDNA sequence. Therefore, these data suggest that a serine protease of the S8A subfamily with specific biochemical properties is involved in senescence-associated protein degradation.

Keywords: 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride; AEBSF; DMSO; DTT; EDTA; EGTA; EP; EP3 protease; Leaf senescence; MS; N-p-toluenesulfonyl benzene propionyl chloride methyl ketone; PAGE; PMSF; PVP; SDS; Subtilisin-like serine protease; TPCK; Triticum aestivum L. cv. Yangmai 3E-158; Wheat; dimethyl sulfoxide; dithiothreitol; endopeptidase; ethylene diamine tetraacetic acid; ethylene glycol bis-(2-amino ether) tetraacetic acid; mass spectrometry; phenylmethanesulfonyl fluoride; polyacrylamide gel electrophoresis; polyvinyl pyrrolidone; sodium dodecylsulfate.