Abstract
金屬硫蛋白III (MT III)是在研究老年癡呆症時，被發現的一種抑制神經 細胞生長的蛋白質。它的分子量很低，含高量的cysteine，並具有與金屬 結合的能力。它的基因已被發現和其它MT的基因相連，並有相同的基因結 構。它的表現只限於腦部，雖然和MT I、MT II 的結構非常類似，但後兩 者卻沒有抑制神經細胞生長的作用，而MT III也不會被鎘、鋅、內毒素 (endotoxin)、類脂醇 (dexamethasone)、等所誘導。到目前為止，對MT III的作用機制還不清楚，為了對它的生物功能進行進一步的探討，我們 選擇了體積較大且取得較方便的豬腦作為研究的材料。首先以PCR 大量複 製豬腦中的 MT III cDNA，再利用所得的產物做為探針對cDNA library進 行篩選。篩選到的cDNA無論是核酸序列或是所推測的胺基酸序列，都與其 它動物的MT III非常類似。我們也利用北方轉漬法 (Northern blot)和 RT-PCR來觀察MT III 的表現情形，結果發現在大腦、小腦、橋腦、延腦 部份都有表現。我們接著利用分子篩和離子交換樹脂等管柱，自豬腦中純 化出一個耐熱、含金屬離子、小分子量的蛋白質，它的pI值為 4.1。經過 比對豬肝所抽出的MT I和 MT II自DEAE管柱中流出的位置，證明我們所純 化出的蛋白質為MT III。同時我們也建立了大量純化的步驟，以利取得大 量的MT III來進行未來的研究。 Metallothionein III (MT III) was first found in the study of Alzheimer's disease (AD). It is a metal-binding, cysteine -rich protein which contained 68 amino acids in human. It shows a growth inhibitory effect on the cortical neuron. The genes encoding MT III are closely linked to other functional MT isogenes. The expression of MT III gene appears to be restricted in brain and fails to respond the induction of Zn, Cd ，bacterial endotoxin and dexamethasone. The mechanism of MT III in inhibiting the neuron growth and its physiological significance are still not clear. In order to study the relationship between the function and structure of MT III, we take the first step by studying the porcine MT III due to the convenience of acessibility of the brain. We conduct Northern blot and RT-PCR analyses, it is found that MT III is ubiquitously expressed in the region of porcine brain we examined. We further purified the MT III protein by the combination of gel-filtration and DEAE ion-exchange chroma- tographies. The pI of the protein was determined to be 4.1. Comparing the protein elution profile on DEAE chromagraphy with porcine liver MTs, we believe that the protein we iso- lated is indeed MT III. We further established the procedures for preparing of MT III in larger quantity for future study.