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研究生:蔡溥仁
研究生(外文):Pui-Jen Tsai
論文名稱:線蟲捕捉菌Arthrobotrysdactyloides捕捉機制之探討(一)鈣調素結合磷酸激化蛋白酵素之選殖(二)油脂親合性三位元G蛋白之生化鑑定
論文名稱(外文):(1) Molecular Cloning of Calmodulin-Binding Protein Kinase and (2) Biochemical Evidence of Heterotrimeric G Protein in the Nematode-Trapping Fungus Arthrobotrys dactyloides
指導教授:杜鎮杜鎮引用關係
指導教授(外文):Jenn Tu
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:110
中文關鍵詞:鈣調素磷酸激化蛋白酵素三位元G蛋白線蟲捕捉菌訊息傳導
外文關鍵詞:calmodulinprotein kinaseheterotrimeric G proteinnematode-trapping fungussignal transduction
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鈣調素結合磷酸激化蛋白酵素為373胺基酸所組成,其基因乃選殖自線蟲捕捉菌Arthrobotrys dactyloides。從現有的基因庫中比較相似胺基酸序列結果發現,本研究所選殖的蛋白質與植物以及人類中所發現的鈣調素結合磷酸激化蛋白酵素極相似,並且還具備有serine/ threonine磷酸激化蛋白酵素的特徵。更進一步嘗試將此選殖基因表達於大腸桿菌中,發現表達的蛋白質具有與鈣調素結合之能力並且此現象可以受鈣離子所調節。從刪減此蛋白質特定位置的研究中,得知自296至324胺基酸序列是扮演著與鈣調素結合的角色,並且以相等比例(1:1)的分子數量形成穩定的複合體。 鈣調素結合激化蛋白酵素是以單一基因的狀態存在於線蟲捕捉菌Arthrobotrys dactyloides中,因此推論此磷酸激化蛋白酵素為一個重要的鈣離子訊息媒介因子。另外在此蛋白質序列中還發現activation loop的存在,經由在threonyl-188位置的磷酸化作用以提高激化蛋白質的酵素活性。一個50 kDa的蛋白質鑑定為鈣調素結合磷酸激化蛋白酵素的催化受質,但此酵素催化作用卻不受鈣離子所調節,推論原因可能是添加過飽和磷酸激化蛋白酵素於酵素反應中,而導致酵素活性的持續地表現。綜合上述結構與生化特性的觀察結果,認為線蟲捕捉菌Arthrobotrys dactyloides的鈣調素結合磷酸激化蛋白酵素是相近似於人類鈣調素結合磷酸激化蛋白酵素的第一群。
此外,在本研究中另已開發出可鑑定油脂親和性三位元G蛋白的全新方法。其原理乃利用百日咳毒素對其放射性受質的酵素催化能力以及G蛋白專一性抗體免疫沉澱法的合併使用。其結果測得一個40 kDa蛋白質即是油脂親和性三位元G蛋白的α次級單元。試驗得知添加GTP-γ-S會減緩百日咳毒素的酵素催化作用,使40 kDa蛋白質的放射性訊號減低。而根據以上證據顯示油脂親和性三位元G蛋白是存在於線蟲捕捉菌Arthrobotrys dactyloides之中,並且可能參與了捕捉機轉的訊息傳遞。

A Ca2+/calmodulin-binding protein kinase (FCaMK) gene was cloned and characterized from Arthrobotrys dactyloides. The cDNA clone contains an open reading frame coding for a protein of 373 amino acids. The deduced structure of FCaMK contains a catalytic domain followed by a calmodulin-binding domain. The amino-terminal region of FCaMK contains all 10 conserved subdomains characteristic of serine/threonine protein kinase. As compared with other calmodulin-related protein kinases, it was observed that the calmodulin-binding region in FCaMK was homologous (32.25%) to those of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) and Ca2+/calmodulin-dependent protein kinase type IV. Additionally, the Escherichia coli-expressed FCaMK presented to bind to its regulator protein calmodulin in a Ca2+-dependent manner. And studies with deletion mutants, the binding ability to calmodulin was unavailable while the FCaMK mutant lacking amino acid residues 296-324. Alternative evidence posed that the Ca2+-regulated protein calmodulin is able to bind to FCaMK. A synthetic peptide designed from the residues 297-324 of FCaMK enabled forming a stable complex with a 1:1 stoichiometry. Additionally, a single copy of encoding fcamk gene was detected by southern blot analysis. It revealed that fcamk is a vital gene and addresses a Ca2+ signaling. The segment of activation loop in the protein kinase FCaMK was identified and up-stream protein kinases might mediate through phosphorylating the threonyl-188 to stimulate the kinase activity. Phosphorylating a 50-kDa substrate which was extracted from Arthrobotrys dactyloides, triggered by FCaMK, it seems to be that the substrate phosphorylation event is independently in the matter of Ca2+ ions. The possible reason may be that the protein kinase FCaMK was over-saturation in all the reactions, while leading to the presence of consecutive kinase activity. Based on these observed data from the structural and biochemical properties, they indicate that the FCaMK belongs to a member of CaMKI subgroup and plays a unique role in Ca2+ signaling in the nematode-trapping fungus Arthrobotrys dactyloides.
A novel methodology for identifying heterotrimeric G protein was established. The method used [32P]ADP-ribosylation catalyzed by pertussis toxin and immunoprecipitation by anti-Gα antiserum or anti-Gβ antisera to trace and identify a 40 kDa polypeptide. The appearance of this [32P] ADP-ribose-labeled polypeptide was also reduced in the presence of GTP-γ-S, a nonhydrolyzed GTP analog. Data indicate that heterotrimeric G protein exists in A. dactyloides and might be involved in the process of cell inflation.

TEXT CONTENTS
1 INTRODUCTION 1
1.1 General view of nematophagous fungi 1
1.2 The nematode-trapping fungus Arthrobotrys dactyloides 2
1.3 The inflating process of constricting rings 2
1.4 The inflating hypothesis of constricting rings 3
1.5 The trapping signals of Ca2+ and GTP-binding protein 4
2 MATERIALS and METHODS 6
2.1 Culture of fungal material 6
2.2 Genomic DNA isolation 6
2.3 Total RNA isolation 7
2.4 Purification of poly(A)+ mRNA 7
2.5 The ZAP-cDNA synthesis 9
2.6 DNA packaging 11
2.7 Reverse transcription and PCR cycling 12
2.8 Random primed DNA labeling 13
2.9 Screening cDNA library 13
2.10 Southern blotting 15
2.11 Excision of phage DNA 16
2.12 Preparation of ZAP phage lysates and isolation of ZAP DNA 17
2.13 FCaMK deletion mutants 18
2.14 Preparation of bacterial sonicates 19
2.15 Activation of Calmodulin-Immobilized Agarose 20
2.16 Calmodulin binding assay 20
2.17 Extraction of total fungal proteins 21
2.18 Assay of kinase activity 22
2.19 Calmodulin/pepide complex detection on nondenaturing PAGE 22
2.20 Determination of protein concentration 23
2.21 Microsomal membrane preparation 23
2.22 ADP-ribosylation by pertussis toxin 23
2.23 Immunoprecipitation 24
3 RESULTS 26
3.1 Cloning of calmodulin-binding protein kinase in A. dactyloides; FCaMK 26
3.2 Alignment of the predicted amino acids of FCaMK with CaMKs 28
3.3 Comparison of the phosphorylated site in activation loops 29
3.4 Comparison of amino acid sequence of calmodulin-binding domain 30
3.5 Deletion studies of amino acid residues 296-324 in FCaMK 30
3.6 A synthetic peptide and its calmodulin binding affinities 38
3.7 Catalytic activities of the protein kinase FCaMK 38
3.8 Molecular assay of copy number in the gene fcamk 39
3.9 A 32P-labeled 40 kDa Gα polypeptide 39
4 DISCUSSION and CONCLUSION 44
4.1 Machinery regulation of the kinase activity: a serine/threonine kinase 44
4.2 Machinery regulation of the kinase activity: calmodulin-binding motif 44
4.3 Machinery regulation of the kinase activity: activation loop 45
4.4 Machinery regulation of the kinase activity: autophosphorylation and
pseudosubstrate 46
4.5 Machinery regulation of the kinase activity: hypothesis 47
4.6 Heterotrimeric G protein 50
FIGURE CONTENTS
Figure 1 Nucleotide and deduced amino acid sequences of FCaMK 27
Figure 2 Domain structure of the FCaMK 32
Figure 3 Comparison of activation loops 34
Figure 4 Comparison of amino acid sequences of calmodulin-binding domains 35
Figure 5 The helic wheel projection of FCaMK 36
Figure 6 Deletion studies of the FCaMK calmodulin-binding domain 37
Figure 7 A purified synthetic peptide forms a stable complex with calmodulin 40
Figure 8 The catalytic acitivities of the protein kinase FCaMK 41
Figure 9 Southern blot of Arthrobotrys dactyloides genomic DNA digested with
various restriction enzymes and probed with the fcamk clone 42
Figure 10 Pertussis toxin (PTX) catalyzed ADP-ribosylation of a 32P-labeled 40 kDa
Gα in membrane proteins immunoprecipitated with G-protein antisera 43
TABLE CONTENT
Table 1 FCaMK and mammalian Ca2+/calmodulin-dependent protein kinases 49
APPENDICES
Appendix 1
The signal transduction pathway in a constricting ring
Appendix 2
Cloning of a Ca2+/calmodulin-dependent protein kinase from the filamentous Arthrobotrys dactyloides (notice of acceptance and author(s) offprint order)

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