牦牛乳酸脱氢酶-1两种遗传变异体的纯化及酶学特性比较

doi:10.3864/j.issn.0578-1752.2009.03.037

中国农业科学 ›› 2009, Vol. 42 ›› Issue (3): 1047-1052 .doi: 10.3864/j.issn.0578-1752.2009.03.037

牦牛乳酸脱氢酶-1两种遗传变异体的纯化及酶学特性比较

西南民族大学生命科学与技术学院

收稿日期:2008-06-16 修回日期:2008-09-22 出版日期:2009-03-10 发布日期:2009-03-10
通讯作者: 赵兴波

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

摘要/Abstract

摘要：

【目的】从乳酸脱氢酶（LDH）水平上探索牦牛(Bos grunniens)低氧适应性分子机制。【方法】利用聚丙烯酰胺凝胶电泳分析牦牛组织中LDH同工酶谱;用比色法测定牦牛、黄牛和水牛心脏、肝脏和肌肉组织中LDH总活力;采用染料亲和层析和DEAE-Sephadex离子交换层析从牦牛心肌组织中纯化LDH1（由4个H亚基组成）的2种遗传变异体，进行酶学性质的比较。【结果】电泳方法检测发现牦牛LDH1存在2种遗传变异体，根据电泳迁移率分别命名为快型和慢型（LDH1-F和LDH1-S）。获得纯化的牦牛LDH1-F和LDH1-S，比活力分别为21.4 U?mg-1蛋白和17.8 U?mg-1蛋白，在SDS-PAGE和PAGE上均显示1条区带。2种变异体以NADH为底物的米氏常数（Km）值差异不大，均显著高于普通牛的LDH1;以丙酮酸钠为底物的Km值LDH1-F小于LDH1-S。试验进一步比较了携带不同LDH1变异体的牦牛心脏、肝脏和肌肉组织中LDH总活力和各种同工酶谱，未见显著差异，而牦牛心脏、肝脏和肌肉组织中LDH总活力显著或极显著低于黄牛和水牛。【结论】牦牛LDH1 2种遗传变异体的Km值存在差异，且Km(NADH)高于普通牛;牦牛心脏、肝脏和肌肉组织中LDH总活力低于普通牛。

关键词: 牦牛, 乳酸脱氢酶, 遗传变异体, 低氧耐受, 分子适应性

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

. 牦牛乳酸脱氢酶-1两种遗传变异体的纯化及酶学特性比较[J]. 中国农业科学, 2009, 42(3): 1047-1052 .

. Purification and Enzyme Property Comparison of Two Genetic Variants of Yak Lactate Dehydrogenase-1
[J]. Scientia Agricultura Sinica, 2009, 42(3): 1047-1052 .

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牦牛乳酸脱氢酶-1两种遗传变异体的纯化及酶学特性比较

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

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