牦牛乳酸脱氢酶A的分离纯化、酶学性质及其基因的克隆

Purification and Property of Yak Lactate Dehydrogenase-A and Sequence of Its cDNA

【目的】分离纯化牦牛骨骼肌中的乳酸脱氢酶-A(LDH-A),并克隆其cDNA。通过比较牦牛与普通牛的LDH-A的酶学性质和cDNA序列,为研究牦牛适应高海拔、低氧环境,在分子水平上找到一些线索。【方法】采用染料亲和层析和DEAE-Sephadex离子交换层析等方法,分别从牦牛和普通牛骨骼肌中纯化LDH-A,并对其酶学性质进行比较;通过RT-PCR方法克隆牦牛LDH-A的cDNA序列,并与GenBank登录的普通牛LDH-A的cDNA序列进行比对。【结果】纯化的牦牛LDH-A比活力为103.9U·mg-1蛋白,纯化倍数为18.2。SDS-PAGE和PAGE分析均显示一条带。酶动力学参数测定显示,牦牛LDH-A的KmNADH为0.097,Km丙酮酸钠为1.897,均不同程度高于普通牛,其中对丙酮酸钠的Km大约是普通牛的2倍。根据牦牛LDH-A的cDNA序列预测的氨基酸序列与普通牛LDH-A的氨基酸序列比较,只有2个氨基酸残基的改变(257Val-Ala和315Tyr-Cys)。【结论】牦牛LDH-A对丙酮酸的高Km可避免骨骼肌中产生过多的乳酸,是适应进化的结果;这种升高可能与其氨基酸序列变化导致的空间结构微小改变有关。

[ Objective ] In order to investigate the molecular mechanism of the ability of yak （Bos. grunniens） to adapt to high altitude and low oxygen environment, the lactate dehydrogenase A （LDH-A） from yak skeletal muscle was purified and characterized and its cDNA was amplified, cloned and sequenced. The kinetics of the yak LDH-A and its cDNA sequence were compared with those of bovine LDH-A. [ Method ] Dye affinity chromatography and DEAE-Sephadex ion-exchange chromatography were used to purify the LDH-A from skeletal muscle of both yak and bovine. The properties of these enzymes were analyzed by kinetic analysis and compared between the two species. The cDNA of yak LDH-A was cloned by reverse transcriptase （RT）-PCR method and compared with the sequence of the bovine LDH-A cDNA in the GenBank. [Result] The yak LDH-A was enriched by 18.2 fold and its relative activity was 103.9 U·mg^-1 protein. Only one band was observed when the purified LDH-A was separated on SDS-PAGE or non-denatured PAGE gel. Kinetic analysis showed that the Michaelis constant （Kin） values of yak LDH-A for NADH and pyruvate were 0.097 and 1.897, respectively, both were higher than that of bovine. In particular, the Km value for pyruvate was about two folds of that of bovine LDH-A. The comparison of cDNA sequences of yak and bovine LDH-A revealed two amino acid replacements （257Val-Ala and 315Tyr-Cys）. [ Conclusion ] The higher Km of yak LDH-A for pyruvate can prevent from producing higher amount of lactate in skeletal muscle that may provide an advantage to adapt to high altitude and low oxygen environment. The two amino acid replacements between yak and bovine LDH-A protein sequences may result in minor conformational change, thus contributing to the kinetic difference.