2007-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/678628摘要：轉穀胺醯氨&#37238;摧化之反應基本上是以酵素Cys 的thiol group 去攻打Gln 上的carbonyl group，NH3&#63978;開，形成Thioester bond。而此Thioester bond 大多是接受Lys 的ε-amino group 攻擊形成Isopeptide bond 而酵素&#63978;開。此Isopeptide bond 亦可接受另一個一級氨、水分子或者醇&#63952;的攻擊。因此此酵素作用的反應為Transadmidation 造成蛋白質之聚合、Amine incorporation 造成polyamines共價結合上蛋白質(polyamine conjugation)、Esterification 造成Gln 轉換成酸酯、Deamidation 造成Gln 轉換成Glu、及Isopeptide cleavage 造成聚合蛋白質的分開。其主要功能在於腫瘤形成及轉移、凝血、瘍口癒口、調控細胞自戕死亡、&#64025;經傳導物質釋放、訊息傳導及細胞間質形成等。轉穀胺醯氨&#37238;之功能必需透過受質之鑑定以及Transamidation 造成之影響&#12458;得以瞭解。本實驗室以新穎方法&#63789;純化及鑑定轉穀胺醯氨&#37238;之受質，活化小鼠(>10 周齡)之睪丸及肝臟其轉穀胺醯氨&#37238;酵素活性，並純化出受質，通過質譜分析鑑定受質的身分，最後以免疫轉漬法以確認受質之真正身分。在鑑定受質中，大概屬下列性質蛋白；細胞骨架及其調節蛋白、Chaperones 及Co-chaperones、內質網蛋白、細胞Detoxification用蛋白、蛋白質轉譯調節蛋白等，大多是與細胞壓&#12459;反應有關之蛋白。其中多個受質也以in vitro Transamidation assay証實是轉穀胺醯氨&#37238;之真正受質。<br> Abstract: Transglutaminases (TG) are Ca2+-dependent enzymes which catalyze a post-translational modification of proteins. The enzyme reaction leads to the formation of an isopeptide bond either within or between polypeptide chains. The γ-glutamyl-ε-lysine crosslinks are formed between the γ-carboxamide group of peptide-bound glutamine residues and the ε-amino group of peptide-bound lysine residues. Polyamines can replace lysine residue in the transamidation reaction in vitro and in vivo. In order to understand the physiological functions of tissue transglutaminase (tTG or TG2), one needs to identify the acyl donor and the acyl acceptor substrates in the transamidation reaction. To this end, we have identified over 70 potential transglutaminase substrates from mouse liver extract and over 100 potential substrates from mouse testis extract by substrate purification and tandem Mass Spectrometry analysis. Five groups of substrates were disclosed; cytoskeleton proteins, proteins involved in ER stress response, molecular chaperones, proteins involved in redox regulation and proteins involved in stress-induced translation arrest. Most of the tTG substrates are intracellular proteins with functions related to cellular response to stress. Importantly, many of the tTG substrates have not been reported. Some of these identified substrates were confirmed by immunoblotting and in vitro transamidation assay.轉穀胺醯氨&#37238細胞壓&#12459反應受質蛋白質轉譯transglutaminaseheat shockstress responseprotein translation