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

• Published in: Phytochemistry (Oxford) 2013-11, Vol.95, p.118-126
• Main Authors: Wang, Renxian, Liu, Shaowei, Wang, Jin, Dong, Qiang, Xu, Langlai, Rui, Qi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; ammonium sulfate; anion exchange chromatography; Cellular Senescence; Cloning, Molecular; complementary DNA; Darkness; DNA, Complementary; Egtazic Acid - pharmacology; enzyme activity; EP3 protease; ethylene glycol tetraacetic acid; gel electrophoresis; Hot Temperature; Leaf senescence; leaves; mass spectrometry; Molecular Sequence Data; nucleotide sequences; Plant Leaves - enzymology; Plant Leaves - physiology; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - metabolism; protein degradation; proteinase inhibitors; Proteolysis; senescence; Serine Endopeptidases - chemistry; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - pharmacology; serine proteinases; Stress, Physiological; Subtilisin - metabolism; Subtilisin-like serine protease; Sulfones - pharmacology; thermal stability; Triticum - chemistry; Triticum - enzymology; Triticum - physiology; Triticum aestivum L. cv. Yangmai 3E-158; Wheat
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2013.06.025

A stable senescence related metal-dependent serine protease of the S8A family with specific physical and chemical properties in dark-induced senescent wheat leaves was purified, identified, and characterized. The figure shows the alignment of EP3 protease with serine proteases in other species. The bold lines indicate the peptide fragments identified by MS/MS. Asterisks identified active site residues of this EP3 protease. [Display omitted] •A stable senescence-related protease EP3 in wheat leaves was purified.•EP3 protease was stable to heat and to some detergents, reducing agents, and organic solvents.•Inhibitor assay indicated that EP3 protease was a metal-dependent serine protease.•EP3 is a subtilisin-like protease as shown by mass spectrometry. 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.