Purification and biochemical characterization of phospholipase A2 from dromedary pancreas |
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Authors: | Bacha Abir Ben Gargouri Youssef Bezzine Sofiane Mejdoub Hafedh |
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Institution: | Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, 3038 Sfax, Tunisia. |
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Abstract: | 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. |
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