赤子爱胜蚓胆碱酯酶的纯化和性质

PURIFICATION AND CHARACTERIZATION OF CHOLINESTERASE FROM EISENIA FOETIDA

赤子爱胜蚓的TritonX－100抽提液经PEG2000-磷酸钾缓冲液处理、离子交换层析及凝胶过滤后，获得电泳纯的胆碱酯酶．纯酶经鉴定为糖蛋白，凝胶过滤法和SDS-PAGE法测得其Mr分别为59000和58000．酸性氨基酸含量约占24％，碱性氨基酸含量约占12％，最大紫外吸收入λ＝272um；酶作用最适温度θ＝39℃，热稳定性较差；最适pH为7．8，在pH7．0～8．0酶较稳定．对碘化硫代乙酰胆碱的Km值为88μmol／L，有过量底物抑制现象．Mg2＋、Ca2＋和Mn2＋对酶活性的影响较小，DFP则对该酶有强烈抑制作用．

Cholinesterase from the Triton X-100 extract of Eisenia foetida was purified to electrophoretic homogeneity by extraction with PEG 2 000/potassium phosphate buffer, ion-exchange chromatography and gel filtration. The purified enzyme was found to be a kind of glycoproteins. The Mr determined by gel filtration was 59 000 and by SDS-PAGE was 58 000. The contents of acidic amino acid and basic amino acid were about 24% and 12%, respctively. Ultraviolet spectrum showed a maximum absorption at 272 um. The enzyme with optimum temperature of 39℃ was unstable when heated. It exhibited optimum activity at pH 7. 8 and kept stable between pH 7. 8 ~ 8. 0. The Michaelis constant for acetylthiocholine iodide was 88 μmol/L. The enzyme activity affected slightly by Mg2+,Ca2+ and Mn2+ was inhibited by excess substrate and strongly by DFP.