2007 Fiscal Year Final Research Report Summary
Degradation mechanism of the chloroplast protein in the cell death of the plant
| Project/Area Number |
18570124
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Shizuoka University |
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Principal Investigator |
AMANO Toyoki Shizuoka University, Department of Biological Science, Associate Professor (90297945)
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| Project Period (FY) |
2006 – 2007
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| Keywords | enzyme / protease / 品質管理 / 発現系 / ATPase / プロテアーゼ / 酵素 / 調節 |
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| Research Abstract |
Plant FtsH pretence is a membrane-associated protease localized on the thylakoid membrane. Its function is to remove a damaged protein from large protein complex. Major physiological substrate of this protease is Dl protein whose subunit plays a key role in the photosystem II. Knockout mutants of this protease shows variegated phenotype in Arabidopsis, therefore physiological function of this protease is to maintenance the chloroplast proteins. In this study, we constructed over-expression system of ATPase and potease domain in FtsH potease from Arabidopsis and tobacco.
In ease of protease domain from tobacco, protein was produced as inclusion body, though them were possible to be activated by urea mediated refolding. Expressed protein showed protease activity on FITC (fluorescein isothiocyanate)-labeled casein. FITC-BSA. (bovine serum albumin) did not digested by this protease, thus substrate specificity was detected on this protease. Biochemical study revealed that protease activity w
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as enhanced by higher concentration of magnesium ion. Optimum pH was determined as alkalescent condition.
ATPase domain in FtsH protease from tobacco was constructed over-expression system in E.coli. This system produced the recombinant protein in the soluble fraction. The protein was purified by Ni^(2+) affinity chromatography. The eluted fraction was verified as ATPase domain by western blotting and peptide sequencing. ATPase activity was obviously detected. Using this system, we analyzed the dependencies of pH, divalent cations, ATP concentration, and the effect of detergents.
Over-expression system for ATPase domain from Arabidopsis was also constructed. Purification was performed using Ni-NTA column. The quality of this protein was verified by western blotting and peptide sequencing. We examined biochemical analysis on the purified protein. And we revealed pH optimum and dependency of Mg^(2+) concentration. We also investigated effect of inhibitors. Among them, EDTA was the highest inhibitory effect. The results suggest that ATPase domain in the FtsH protease depends on divalent cations. We are trying to determine Km and Vmax, optimum temperature, and effective substrate except for ATP. Less
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