- 學位論文
芋頭半胱胺酸蛋白酶抑制因子上不同區域抑制特性分析
Investigation on inhibitory characterization of different segments of tarocystatin from Colocasia esculenta
摘要
Tarocystatin屬於cysteine proteinase inhibitor中的一員，由CeCPI基因大量表現且儲藏於芋頭(Colocasia esculenta)塊莖中。Tarocystatin胺基酸序列含有一個高度保留的N端序列，與水稻OC-Ⅰ相似(~97% identity)，並在C端有一段廣泛延伸的序列。本實驗利用大腸桿菌表現蛋白的系統，分別表現CeCPI全長、N端及C端的蛋白片段並進行一系列的生化及生理活性分析。全長及N端片段都有抑制papain的活性，而C端卻相反的能活化papain的活性。為了清楚其抑制特性是否不同，利用Lineweaver-Burk plot 法分析其抑制行為。全長的tarocystatin遵守混合型抑制行為(Kia=0.098 μM，Kib=0.252 μM)，N端為競爭型抑制行為(Ki=0.057 μM)，而C端為異位活化作用。分析其不同受質濃度下對papain的平均抑制活性，全長與N端的平均抑制活性分別為55%及39%，而C端卻具有18%活化papain的活性，約相當於全長與N端抗性之間的差距(16%)。因此不排除C端序列在第二群的phytocystatin中可能扮演調節目標蛋白構形的角色。而N端功能則類似OC-Ⅰ，提供抑制劑競爭活性區的能力。本實驗結果可推論全長對papain之抑制能力較N端片段具有較高之活性，且對papain具較高的專一性 (因其較不易對真菌產生抗性)及較長的半衰期，適合當作防禦性蛋白，且分子量較大比僅N端片段更適合當作儲藏性蛋白。
並列摘要
Tarocystatin (CeCPI), a group-2 cysteine proteinase inhibitor in planta, is a defensive protein against phytopathogenic nematodes and fungi. This protein is made up of 205 amino acids, including N terminal segment (Nt peptide) of 98 amino acids and C terminal segment (Ct peptide) of 107 amino acids. The full length (FL), Nt peptide and Ct peptide segments of tarocystatin from Colocasia esculenta (Kaohsiung No.1), were separately amplified by PCR and expressed as GST fusion proteins in E. coli. Kinetic analysis of FL, Nt peptide and Ct peptide on papain activity revealed that FL exhibited mixed type inhibition (Kia =0.098 µM and Kib =0.252 µM) and Nt peptide showed competitive inhibition (Ki, 0.057 µM), whereas Ct peptide enhanced papain activity. Moreover, FL exhibited stronger antipapain activity than Nt peptide. But the antifungal activity of Nt peptide appears to be greater than that of FL, and Ct peptide did not show any antifungal activity indicating that antifungal effect is not related to proteinase inhibition. A shift in the inhibition pattern from competitive inhibition of Nt peptide segment alone to mixed type inhibition of FL implied that Ct peptide has got an influence on FL function, and Nt peptide can determine the fate of CeCPI function. Based on the inhibitory kinetics of FL and fragments of CeCPI on papain activity, a model for group-2 phytocystatin inhibitory mechanism has been proposed. The physiological significance for two varied types of proteins is also discussed.
參考文獻
延伸閱讀
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