VILIP-1 已知可調控 Guanylyl cyclase-B (GC-B) 活性，增加細胞內 cGMP
濃度而 VILIP-3 則否。 本論文主要目標係探討 VILIP-1 調控 GC-B 活性
的因子，因此表現重組蛋白 Myristoylated 與 Nonmyristoylated VILIP-1、
VILIP-3、Chimeric VILIPs 和 Mutant VILIP-1，重組蛋白以離子交換層析與
分子篩 (Gel filtration) 方式進行純化。 Circular Dichroism 分析顯示
VILIPs 主要二級結構為 α-helix ，而以蛋白質酶水解方法及螢光分析證實
Myristoylation、Ca2+ 和Mg2+ 等均會影響 VILIPs 結構且會誘導不同的構形
變化。 分子篩結果顯示 VILIP-1 的 EF-hand 3&4 及 Myrstoylation 與
VILIP-1 的 Dimerization 有關，而 Dimer 與 Monomer 呈現動態平衡。 以
猪腦組織細胞膜與 Liposome binding assay 結果顯示 Myristoylation、Ca2+
和Mg2+ 會增加 VILIPs 與它們的結合能力。 In vitro guanylyl cyclase
activity assy 結果顯示 Myristoylation 和 VILIP-1 的 EF-hand 1 參與調控
GC 活性，而此作用需要Mg2+或Ca2+ 的存在。 以上結果證實 VILIP-1 的
Myristoylation 及EF-hand 1 在 VILIP-1 調控 GC 活性上具有重要密切關
係。 此調控作用與 Ca2+ 或 Mg2+ 誘導產生構形之變化有關，而增加
VILIP-1 結合至細胞膜上以協助 VILIP-1 調控 GC 活性； 但與
Oligomerization 狀態Dimerization 沒有相關性。

It has been well-known that VILIP-1 but not VILIP-3 regulates the activity of
guanylyl cyclase-B. In order to identify the modulated region within VILIP-1
on regulating guanylyl cyclase-B activity, the recombinant myristoylated and
nonmyristoylated VILIPs (VILIP-1, VILIP-3, chimeric VILIPs, and mutant
VILIP-1) were prepared in the present study. The recombinant proteins were
purified using ion-exchanger chromatography followed by gel filtration. CD
spectra indicated that the secondary structure of VILIPs was dominant with
α-helix, reflecting a well-conserved EF-hand structure. Tryptic digestion assay
and the fluorescence measurement showed that myristoylation, Ca2+ and Mg2+
differently induced the conformational changes of VILIPs. The results of gel
filtration chromatography reflected that the EF-3&4 of VILIP-1 and
myristoylation were involved in the dimerization of VILIP-1, and the dimer and
monomer were converted each other in a dynamic manner. The porcine brain
membrane binding assay and liposome binding assay showed that the binding
capability of VILIPs were markedly enhanced by myristoylation, Mg2+ and Ca2+.
Myristoylation and the intact EF-1 of VILIP-1 were found to essential for
the regulation of guanylyl cyclase activity in the presence of Mg2+ and Ca2+.
Taken together, theses results suggest that myristoylation and EF hand-1 of
VILIP-1 are the structural elements crucial for regulating the guanylyl cyclase
activity. In contrast to oligomerization of VILIP-1, Mg2+ and Ca2+ -induced
conformational changes of VILIP-1 and enhancement of the binding of VILIP-1
with membrane by Mg2+ and Ca2+ partly but not heavily involve in the action.

中文摘要 ----------------------------------- i
英文摘要 ----------------------------------- ii
英文縮寫 ----------------------------------- 1
序言 --------------------------------------- 1
實驗材料 ----------------------------------- 4
實驗方法 ----------------------------------- 6
結果 --------------------------------------- 15
討論 --------------------------------------- 20
參考文獻 ----------------------------------- 23
表 ----------------------------------------- 29
圖 ----------------------------------------- 32

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