关键词: 牦牛, 乳酸脱氢酶, 纯化, 动力学性质, 基因克隆

Abstract: [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.

Key words: Yak, lactate dehydrogenase, purification, gene cloning