巴西橡胶树胶乳磷脂酶A2的分离纯化及部分酶学性质鉴定

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

关键词:  巴西橡胶树  磷脂酶A₂  酶的纯化  酶的性质

Key words:  Hevea brasiliensis  phospholipase A₂  purification of enzymes  enzymological properties

摘要:

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K_m和V_max。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43 kDa。最适反应温度为37 ℃,最适反应pH为8.0,K_m为0.44 mmol·L^-1,V_max为7.22 μmol·(mL·min)^-1。最适Ca²⁺浓度为50 μmol·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。

Abstract:

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 K_m,V_max,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 K_m was 0.44 mmol·L^-1. The V_max 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.