兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。

Studies on Purification and Properties of Rabbit Muscle Glycerol 3-Phosphate Dehydrogenase

Objective: To explore the purification and properties of glycerol 3-phosphate dehydrogenase(GPDH) from rabbit muscle and provide theoretical support for its applications on enzyme linked diagnosis reagents in vitro. Methods: Glycerol 3-phosphate dehydrogenase from rabbit muscle was purified by ammonium sulfate fractional precipitation, DEAE-sepharose, Blue-sepharose and Hydroxyapatite. Sephacryl S-200HR chromatography and gradient PAGE were used to determine its molecular weight, kinetic properties such as effect of pH, temperature, substrate concentration and chemicals were also determined. Results: The purified enzyme showed a prominent single band on PAGE, and its molecular weight was estimated to be 115～122 kDa. The enzyme was stable below 45℃ and pH 6.0～9.0 with maximal activity at 45℃, pH 9.0. K_m value of the enzyme was calculated to be 7.4×10^-3mol/L for substrate glycerol 3-phosphate, and to be 1.47×10^-4mol/L for substrate NAD⁺. Ba²⁺, Mn²⁺, Fe²⁺, Al³⁺, Cu²⁺, Ni²⁺, Ag⁺, Hg²⁺, NaN₃ and EDTA inhibited the activity of GPDH at various degree, whereas Mg²⁺, Ca²⁺, Co²⁺, Zn²⁺ could stimulate the enzyme activity, NaF had no obvious effect on glycerol 3-phosphate dehydrogenase.