title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Renxian
authors.initials: R
authors.lastname: Wang
authors.forename: Shaowei
authors.initials: S
authors.lastname: Liu
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Dong
authors.forename: Langlai
authors.initials: L
authors.lastname: Xu
authors.forename: Qi
authors.initials: Q
authors.lastname: Rui
mesh_terms: D000595:Amino Acid Sequence; D016922:Cellular Senescence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003624:Darkness; D004533:Egtazic Acid / Q000494:pharmacology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology / Q000502:physiology*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013312:Stress, Physiological; D020860:Subtilisin / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014908:Triticum / Q000737:chemistry / Q000201:enzymology* / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D004533:Egtazic Acid; D012697:Serine Endopeptidases; D020860:Subtilisin
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