兔乳酸脱氢酶C_4的分离纯化及动力学研究

Purification and kinetic study on rabbit lactate dehydrogenase-C_4

目的：建立兔乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）分离纯化的新方法，研究该酶的动力学特性。方法：采用ＢｌｕｅＤｅｘｔｒａｎ和Ｓｅｐｈａｒｏｓｅ４Ｂ交联制成ＢｌｕｅＤｅｘｔｒａｎ－Ｓｅｐｈａｒｏｓｅ４Ｂ染料配体亲和层析柱，结合ＱＡＥ－ＳｅｐｈａｄｅｘＡ５０离子交换层析分离提纯兔ＬＤＨ－Ｃ４，以作图法测定该酶动力学数据。结果：纯化的ＬＤＨ－Ｃ４经ＳＤＳ－ＰＡＧＥ及琼脂糖凝胶电泳证明为单一组分，相对分子质量１３６０００。纯化得率４５．５％，纯化倍数３１６倍。兔ＬＤＨ－Ｃ４动力学测定，其对底物乳酸、丙酮酸的Ｋｍ值分别为１．１１ｍｍｏｌ／Ｌ、０．２３ｍｍｏｌ／Ｌ；产物乳酸、丙酮酸对其均有非竞争性抑制作用，Ｋｉ值分别为９．１１ｍｍｏｌ／Ｌ、０．０２７ｍｍｏｌ／Ｌ。结论：本法是一种简便、快速、高效的分离纯化免ＬＤＨ－Ｃ４新方法；动力学性质表明该酶对丙酮酸的亲和力大于乳酸，且易受产物丙酮酸的抑制。

Objective:To establish a new method to purify rabbit LDH-C4 and investigate kinetic properties of LDH-C4.Methods:LDH-C4 from rabbit testes was separated by Blue Dextran-Sepharose 4B dye ligand affinity chromatography and QAE-Sephadex A50 ion exchange chromatography.Results:The purified LDH-C4 exhibited a single protein band on SDS-PAGE and agarose gel with molecular weight of 136 000daltons.The yield of the purified LDH-C4 in this procedure was 45.5% with a 316-fold purification.Studies on Km for pyruvate,lactate were performed.Km values for substrate pyruvate and lactate were 0.23mmol/L and 1.11 mmol/L, respectively.LDH-C4 were inhibited noncompetitively by lactate or pyruvate and the Ki values were 9.11 mmol/L and 0.027 mmol/L,respectively.Conclusion:The results suggest that this novel method is simple,rapid and effective.The study of kinetic properties demonstrate that the rabbit LDH-C4 exhibits a higher affinity for pyruvate than lactate and the enzyme activity is inhibited by product pyruvate significantly.