牦牛乳酸脱氢酶A的分离纯化、酶学性质及其基因的克隆

 【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103.9 U?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Km NADH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。

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

Objective] In order to study the adaptation of yak (Bos. grunniens) to high altitude and low oxygen plateau at molecular level, lactate dehydrogenase A (LDH-A) from yak skeletal muscle was purified, and LDH-A cDNA was cloned. The kinetics and cDNA of yak LDH-A were compared with those of bovine LDH-A. Methods] Dye affinity chromatography and DEAE-Sephadex ion-exchange chromatography were used to purify LDH-A from skeletal muscle of yak and bovine, and their enzyme properties were compared. The cDNA of yak LDH-A was cloned by RT-PCR methods and compared with that of bovine LDH-A in the GenBank. Result] The relative activity of purified LDH-A was 103.9 U/mg protein, with purification factor of 18.2. Only one band was observed when the purified LDH-A was separated with SDS-PAGE or native PAGE. Kinetic analysis showed that Michaelis constants (Km) value for NADH was 0.097, and Km value for pyruvate was 1.897, both was higher than that of bovine. The Km value for pyruvate of yak LDH-A was about two folds that of bovine. Hg2+ inhibited LDH-A activity, and the inhibition effect was partly removed by adding β- mercaptoethanol. Conclusion] The high Km for pyruvate of yak LDH-A can prevent from producing too much lactate in skeletal muscle, and might be a result of adaptive evolution.The two amino acid replacements are responsible for the increased Km value.