Characterization of cysteine protease induced by oxidative stress in cells of Chlamydomonas sp. strain W80.
Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert-butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K(m) value was estimated to be 44.4 microM for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS-PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30-90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions.
Usui
Masatoshi
M
Department of Biological Science, Faculty of Science, Shizuoka University, 422-8529 Shizuoka, Japan.
Tanaka
Satoshi
S
Miyasaka
Hitoshi
H
Suzuki
Yoshimi
Y
Shioi
Yuzo
Y
D000818:Animals; D002696:Chlamydomonas / Q000201:enzymology*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002734:Chlorophyll / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D018384:Oxidative Stress; D013379:Substrate Specificity
D002734:Chlorophyll; D003546:Cysteine Endopeptidases