Biochemical and molecular characterization of senescence-related cysteine protease-cystatin complex from spinach leaf
| Autor: | Masashi Satoh, Akemi Yamaguchi, Satoshi Hayasaka, Takayuki Tajima, Yuzo Shioi, Katsuhiko Yoshimatsu, Shuhei Matsushima |
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| Rok vydání: | 2010 |
| Předmět: |
Proteases
Physiology Molecular Sequence Data Trimer Plant Science Chromatography Affinity Cysteine Proteases Spinacia oleracea Genetics Amino Acid Sequence Cloning Molecular Polyacrylamide gel electrophoresis Cellular Senescence Phylogeny Plant Proteins Gel electrophoresis Sequence Homology Amino Acid biology Reverse Transcriptase Polymerase Chain Reaction Cell Biology General Medicine Hydrogen-Ion Concentration Cystatins Cysteine protease Molecular biology Enzyme assay Plant Leaves Biochemistry biology.protein Electrophoresis Polyacrylamide Gel Cystatin Cysteine |
| Zdroj: | Physiologia Plantarum. 141:97-116 |
| ISSN: | 0031-9317 |
| DOI: | 10.1111/j.1399-3054.2010.01425.x |
| Popis: | Cysteine proteases (CPs) with N-succinyl-Leu-Tyr-4-methylcoumaryl-7-amide (Suc-LY-MCA) cleavage activity were investigated in green and senescent leaves of spinach. The enzyme activity was separated into two major and several faint minor peaks by hydrophobic chromatography. These peaks were conventionally designated as CP1, CP2 and CP3, according to their order of elution. From the analyses of molecular mass, subunit structure, amino acid sequences and cDNA cloning, CP2 was a monomer complex (SoCP-CPI) (51 kDa) composed of a 41-kDa core protein, SoCP (Spinacia oleracea cysteine protease), and 14-kDa cystatin, a cysteine protease inhibitor (CPI), while CP3 was a trimer complex (SoCP-CPI)(3) (151 kDa) of the same subunits as SoCP-CPI and showed a wider range of specificity toward natural substrates than SoCP-CPI. Trimer (SoCP-CPI)(3) was irreversibly formed from monomers through association. The results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNAs of CPI and SoCP are hardly expressed in green leaves, but they are coordinately expressed in senescent leaves, suggesting that these proteases involve in senescence. Purified recombinant CPI had strong inhibitory activity against trimer SoCP, (SoCP)(3) , which had a cystatin deleted with K(i) value of 1.33 × 10(-9) M. After treatment of the enzyme with a succinate buffer (pH 5) at the most active pH of the enzyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and activity analyses showed that cystatin was released from both monomer SoCP-CPI and trimer (SoCP-CPI)(3) complexes with a concomitant activation. Thus, the removal of a cystatin is necessary to activate the enzyme activity. |
| Databáze: | OpenAIRE |
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