頁籤選單縮合
題名 | Characterization of Phospholipase A[feaf](PLA[feaf])from Taiwan Cobra: Isoenzymes and Their Site-Directed Mutants |
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作者 | Pan,Fu-ming; Chao,Shu-chi; Wu,Shih-hsiung; Chang,Wen-chang; Chiou,Shyh-horng; | 書刊名 | 中央研究院生物化學研究所論文集 |
卷期 | 24 1998[民87.] |
頁次 | 頁97-103 |
分類號 | 361.4 |
關鍵詞 | Phospholipase A[feaf]; PLA[feaf]; |
語文 | 英文(English) |
英文摘要 | EitTacellular and secretory phospholipase A�� (PLA��), a class of phosphoUpid digesting enzyme, is widely distributed in animal venoms of reptiles and insects. Two cDNAs encoding PLA�疳soenzymes from Taiwan Cobra (Naja Ttoja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli. The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA �� with the same molecular masses of native enzymes as determined by mass spectrometry. The recombinant PLA �� was also shown by circular dichroism to possess a secondary structure similar to native PLA ��. The enzymatic activity of the major isoenzyme (PLA �荃澡� ) is higher than the other minor isoenzyme (PLA �荃澡� ), which shows two amino acid difference from PLA �荃澡�. Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA ��, i.e., His-47 and Asp-93. Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity. Similarly, the mutant Asp-93 → Asn (D93N) also retained little activity. These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLAa. Com Puter graphic study, based on homology modelling, highlights the differences between native PLA �� isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity. |
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