Bacha Abir Ben, Gargouri Youssef, Bezzine Sofiane, Mejdoub Hafedh
Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, 3038 Sfax, Tunisia.
Biochim Biophys Acta. 2006 Aug;1760(8):1202-9. doi: 10.1016/j.bbagen.2006.03.014. Epub 2006 Apr 19.
Dromedary pancreatic PLA2 (DrPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat and acidic treatment (70 degrees C; pH 3.0), precipitation by ammonium sulphate and ethanol respectively, followed by sequential column chromatographies on Sephadex G-50, MonoS Sepharose, MonoQ Sepharose and C-8 reverse phase high pressure liquid chromatography. Purified DrPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 13748.55 Da. A specific activity of 600 U/mg for purified DrPLA2 was measured at optimal conditions (pH 8.0 and 37 degrees C) in the presence of 3 mM NaTDC and 7 mM CaCl(2) using PC as substrate. The sequence of the first fourteen amino-acid residues at the N-terminal extremity of DrPLA2 was determined by automatic Edman degradation. One single sequence was obtained and shows a close similarity with all other known pancreatic secreted phospholipases A2.
单峰骆驼胰腺磷脂酶A2（DrPLA2）从脱脂胰腺中纯化得到。经过加热和酸性处理（70℃；pH 3.0）、分别用硫酸铵和乙醇沉淀，随后依次在葡聚糖G - 50、单磺酸琼脂糖、单季铵琼脂糖和C - 8反相高压液相色谱上进行柱色谱分离后，获得了纯蛋白。纯化后的DrPLA2不是糖基化蛋白，是一种分子量为13748.55 Da的单体蛋白。在最佳条件（pH 8.0和37℃）下，以磷脂酰胆碱（PC）为底物，在3 mM十六烷基三甲基溴化铵（NaTDC）和7 mM氯化钙（CaCl₂）存在的情况下，测得纯化后的DrPLA2的比活性为600 U/mg。通过自动埃德曼降解法测定了DrPLA2 N末端前14个氨基酸残基的序列。获得了一个单一序列，该序列与所有其他已知的胰腺分泌型磷脂酶A2具有高度相似性。
