Scientia Agricultura Sinica ›› 2009, Vol. 42 ›› Issue (3): 1047-1052 .doi: 10.3864/j.issn.0578-1752.2009.03.037

• ANIMAL SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Purification and Enzyme Property Comparison of Two Genetic Variants of Yak Lactate Dehydrogenase-1

  

  1. 西南民族大学生命科学与技术学院
  • Received:2008-06-16 Revised:2008-09-22 Online:2009-03-10 Published:2009-03-10
  • Contact: ZHAO Xing-bo

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Abstract:

【Objective】 The aim of the present study was to investigate at the level of the glycolytic enzyme lactate dehydrogenase (LDH) the molecular mechanism by which yak (Bos grunniens) adapts to low oxygen environment. 【Method】 Yak LDH isozymes were assayed by polyacrylamide gel electrophoresis. Total LDH activities in heart, liver and skeletal muscle of yak, yellow cattle and buffalo were analyzed by spectrophotometry method. Dye affinity chromatography and DEAE-Sephadex ion-exchange chromatography were employed to purify lactate dehydrogenase-1 (LDH1, which is composed of four H subunits) genetic variants from yak cardiac muscle in order to compare the enzyme properties. 【Result】 Electrophoresis revealed two genetic variants of yak LDH1. According to the difference of electrophoresis mobility, the fast-band LDH1 was named as LDH1-F and the slow-band as LDH1-S. The relative activity of the purified LDH1-F and LDH1-S were 21.4 U?mg-1 and 17.8 U?mg-1 protein, respectively, and both of them exhibited only one band on SDS-PAGE or native PAGE. Kinetic analysis showed that Michaelis constants (Km) value for NADH existed no difference between the two variants, but significantly higher than that of bovine LDH1. The Km value for pyruvate of LDH1-F was lower than that of LDH1-S. Yaks carrying LDH1-F and LDH1-S variants showed similar total LDH activities and LDH isozyme profiles in heart, liver and skeletal muscles. Total enzymatic activity assay showed that the activities of LDH in heart, liver and skeletal muscles of yaks were significantly lower than those of yellow cattle or buffalo. 【Conclusion】 Results from this experiment indicate that the two genetic variants of yak LDH1 have differences in their Km value, and the Km(NADH) value is higher than that of bovine LDH1. The total LDH activities in heart, liver and skeletal muscles of yaks were significantly lower than that of cattle. These changes in yak LDH1 are attributed to the adaptation to hypoxic environment.

Key words: yak, lactate dehydrogenase, genetic variants, hypoxia tolerance, molecular adaptation

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