硒对华贵栉孔扇贝碱性磷酸酶的酶动力学及理化性质研究

The kinetics and physical & chemical characteristics of alkaline phosphatase from the scallope Chlamys nobilis(Reeve) effected by trace Selenium

探讨了微量硒(Se^(4+))对华贵栉孔扇贝(Chlamys nobilis(Reeve))碱性磷酸酶(AKP)的作用。以催化动力学原理研究了不同缓冲系统中微量硒时AKP的影响。结果表明,Se^(4+)对谈酶的作用与酶所处的环境有关。在0.05 mol/L Na_2CO_3-NaHCO_3(pH 9.0)缓冲溶液中Se^(4+)浓度小于12.5 mmol/L时呈现出激活作用;Se^(4+)浓度大于12.5 mmol/L时呈现出非竞争性的抑制作用,其抑制常数为6.81×10^(-2)mol/L。在0.05 mol/L Tris-HCl缓冲系统(pH 9.0)中,微量Se^(4+)对AKP具有强烈竞争性抑制作用,其抑制常数为3.79×10^(-1)mol/L。经紫外吸收光谱、荧光发射光谱和圆二色谱的实验表明,微量Se^(4+)与AKP作用后,该酶的构象已发生了变化。研究发现海水环境中若含有微量Se^(4+)(SeO_3^(2-)),可以促进扇贝AKP的水解。利于贝类的生长发育和外壳的形成,并有益于贝类的繁殖。

The effects of trace selenium on alkaline phosphatase（AKP） from Chlamys nobilis （Reeve） were estimated. The behaviors of AKPase in different buffer systems were studied by using the principle of enzymatic catalytic kinetics . The results have indicated that the role of Se^4＋ on the AKPase is related to the buffer systems that AKPase existed in. The activation of AKPase was found in 0.05 mol/L Na2 CO3-NaHCO3 （pH = 9.0） system when Se^4＋ concentration is below to 12. 5 mmol/L . And the inhibition of AKPase as noncompetitive inhibition was found when Se^4＋ concentration is over 12.5 mmol/L . The inhibition constant is 4.9 × 10^- 2 mol/L . While the strongly competitive role of AKPase was in 0.05 mmol/L Tris-HC1 buffer system where the trace selenium exsisted. The inhibition constant is 3.9×10^-3mol/L. The effects of trace selenium（SeO3^2- ） on the changes in molecular conformation of AKPase from C. nobilis（Reeve） have been observed, detected by UV-absorption spectra, fluorescence emission spectra and circular dichroism spectra. The results showed that trace selenium which exists in the normal breeding seawater may promote AKPase hydrolysis of scallape, make for the growth of seashell and its crust forming. Therefore, trace seleni- um in seawater should avail to the propagating of seashell. While pollution of the breeding environment, such as variations of the alkalinity and the presence of harmful metal ions and organic solvents, the pollution affects exists the enzyme activity and its conformation which affects the growth and survival of the animal.