巴西橡胶树胶乳磷脂酶A_2的分离纯化及部分酶学性质鉴定

通过丙酮沉淀、DEAE-纤维素离子交换柱层析和Sephadex G-100凝胶过滤柱层析等分离纯化技术,对巴西橡胶树胶乳C-乳清磷脂酶A2进行分离纯化。用SDS-PAGE测定其亚基的相对分子量。测定该酶最适温度和pH,动力学常数Km和Vmax。并测定Ca2+和La3+对酶活性的影响。结果显示:该酶被纯化了49.47倍,产率为5.12%。SDS-PAGE检测为单一条带,其亚基相对分子量约43kDa。最适反应温度为37℃,最适反应pH为8.0,Km为0.44mmol·L-1,Vmax为7.22μmol.(mL.min)-1。最适Ca2+浓度为50μmol·L-1,稀土元素La3+离子对磷脂酶A2活性有抑制作用,但加入Ca2+后可缓解La3+对磷脂酶A2活性的抑制作用。胶乳C-乳清磷脂酶A2与其他植物磷脂酶A2在Ca2+的依赖性上存在差异。研究结果为今后探索橡胶树胶乳磷脂酶A2的催化机理、调节机理及生理功能等奠定了基础。

Purification and enzymatic characterization of phospholipase A2 from latex of Hevea brasiliensis

A phospholipase A2(PLA2)was purified from the latex C-serum of Hevea brasiliensis by acetone precipitation,DEAE-Cellulose ion exchange chromatography,Sephadex G-100 gel filtration. SDS-PAGE was used to determine the Mr of the subunit of PLA2. Then the Km,Vmax,the optimum temperature and pH of PLA2 were tested. The effect of Ca2+ and La3+ on PLA2 activity were assayed. A 49.47-fold purification was obtained with the recovery of 5.12% activity. The results showed that a single band on SDS-PAGE with a subunit Mr of 43 kDa. Latex C-serum PLA2 had an optimum temperature at 37 ℃,and an optimum pH at 8.2. The Km was 0.44 mmol·L-1. The Vmax was 7.22 μmol·(mL·min)-1. The Ca2+ ion optimum concentration for activity was determined to be 50 μmol·L-1. The activity of PLA2 was inhibited by La3+. The inhibited effect of La3+ was palliated after added calcium ion. There are differences of Ca2+ dependence between latex C-serum and other plant PLA2. The results provide a research basis for catalytic mechanism,regulation,and physiology function of PLA2 in latex.