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KMID : 0381120120340010013
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Genes and Genomics
2012 Volume.34 No. 1 p.13 ~ p.18
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Biochemical analysis of a Chinese cabbage phytocystatin-1
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Hong Joon-Ki
Je Ji-Hyun
Song Chi-Eun
Hwang Jung-Eun
Lee Yeon-Hee
Lim Chae-Oh
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Abstract
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The phytocystatins are inhibitors of papain-like cysteine proteinases that are implicated in defense mechanisms and the regulation of protein turnover. BCPI-1, a Brassica rapa (Chinese cabbage) phytocystatin isolated from flower buds, contains an extended C-terminal region that contains a single Cys residue at position 102. In an effort to investigate the role of the C-terminus and this Cys residue in BCPI-1 activity, purified recombinant proteins of BCPI-1, including wild-type BCPI-1 (wtBCPI-1), N-terminus BCPI-1 (BCPI-1ΔC), C-terminus BCPI-1 (BCPI-1ΔN), and BCPI-1 with a single Cys residue exchange to Ser (BCPI-1C102S), were generated and their inhibitory activities against papain were investigated. Kinetic analysis revealed that the monomeric forms of wtBCPI-1 (K i = 6.84 ± 0.3 × 10?8 M) inhibited papain more efficiently than the dimeric forms of wtBCPI-1 (K i = 1.01 ± 0.5 × 10?7 M). Experiments with recombinant BCPI-1C102S demonstrated that the dimerization of wtBCPI-1 caused by the formation of an intermolecular disulfide bond at the cysteine residue. The inhibitory activity of the recombinant proteins, except BCPI-1ΔN, was reduced in the pH range of 7.0?11.5 and was highly stable over a wide range of temperatures. Thus, dimerization mediated by the cysteine residue in the extended C-terminal region and alkaline conditions reduced the inhibitory activity of BCPI-1.
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KEYWORD
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Inhibitory activity, Protein kinetics, Protein stability, Recombinant proteins
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