純化與定性大白鼠腦細胞膜的五核甘酸?

Abstract

五核甘酸?(5’-nucleotidase, 5'NT)是一種細胞膜上的標記酵素(Marker enzyme)，廣泛存在於細胞膜上，其主要的作用機制是將五腺核甘單磷酸(Adenosine 5’-monophosphate)水解成腺嘌呤(Adenosine)和單磷酸(monophosphate)。本實驗是利用介面活性劑Sulphobetaine14 (SB-14)來萃取並純化腦細胞膜上的五核甘酸?，利用兩支以100mM和10mM Tris所平衡的DE-52陰離子交換管柱(Anion exchanger colume)和一支AcA-34膠體過濾管柱(Gel filitraction colume)進行酵素純化。 經過純化所得到的腦的五核甘酸?以SDS-PAGE、自然膠體電泳(Native gel electrophoresis)、和膠體過濾法(Gel filitraction chromatograph)等方法所測得的分子量均在65,000-75,000 Dalton之間，約是同時純化得到的肝臟細胞膜的五核甘酸?(其分子量?150,000 Dalton)的一半。因?肝臟的五核甘酸??雙原分子，故推測腦的五核甘酸?應以單原分子形式存在。 此外，在比較這兩種不同組織的酵素性質時，可發現兩者有很大的差別。例如：腦的5'NT的Km值?12μM，肝臟?20μM；而腦的5'NT的Vmax則?82nm Pi/min，肝臟?151 nm Pi/min。比較兩者的等電點(Isoelectric point, pI)時發現：腦的5'NT的pI值在6.5到8.0之間，而肝臟的5'NT的pI則在4.8到5.2之間。但兩者有相同的作用酸鹼值(Optimal pH)?7.5，並皆可?磷脂?D所作用，表示兩者都是以聚醣磷脂醯肌醇(Glycosyl-phosph-atidylinositol, GPI)接合於細胞膜上。

5’-Nucleotidase (5’NT, EC 3.1.3.5) was extracted from the rat brain membrane by using the zwitterionic Sulphobetaine 14 (SB-14). After two ion-exchange filitraction chrpmatpgraphies the 5’NT was purified with a final specific activity of 8.25 units/mg of protein. Based on the estimations by gel filitraction, native gel electrophoresis sand SDS-PAGE, I concluded the enzyme with a molecular weight of 65 kDa-75 kDa, Its isoelectric point is between 6.5-8.0. The enzyme has a Km value of 12 μM by using 5‘AMP as substrate, and has an optimal enzyme activity at pH 7.5. The treatment with phospholipase D could shift the enzyme to a more basic posit ion in the native gel electrophoresis system, It is concluded that the 5’-nucleotidase is anchored on the membrane of brain with the glycosyl-phosphatidylinositol (GPI).