Reassociation of Lactic Dehydrogenase from Pig Heart Studied by Cross-Linking with Glutaraldehyde
Reassociation of Lactic Dehydrogenase from Pig Heart Studied by Cross-Linking with Glutaraldehyde
复制标题
通过戊二醛交联研究猪心乳酸脱氢酶的重新结合
DOI：
10.1515/znc-1981-9-1013
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
R. Jaenicke
中科院分区：
文献类型：
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作者：
G. Bernhardt;R. Rudolph;R. Jaenicke
摘要戊二醛交联法已成功地应用于寡聚酶（R。赫尔曼河Rudolph和R. Jaenicke Nature 277，243-245（1979））。在本研究中，乳酸脱氢酶的组装从猪心使用这种方法进行了研究。为了消除由过度折叠反应引起的扰动，在0°C下在0.8M Na 2SO 4存在下进行酸解离。在最佳条件下，在不到2分钟的时间内实现了四聚体酶的完全交联。复溶过程中的交联证明二聚体是唯一的复溶中间体。二聚体→四聚体的转变被发现是速率限制的再结合和再活化，这表明四聚体是酶活性物种。重构过程中单体的存在表明四聚体形成之前是快速的单体-二聚体平衡。动力学模型的平衡常数K = 3 ± 1 · 107升·mol-1，二级速率常数k = 1.4 ± 0.2 · 104升· mol-1 · s-1。
Abstract Cross-linking with glutaraldehyde has been successfully applied in order to analyze the kinetics of reassociation of oligomeric enzymes (R. Hermann, R. Rudolph, and R. Jaenicke Nature 277, 243-245 (1979)). In the present study the assembly of lactic dehydrogenase from pig heart is investigated using this approach. In order to eliminate perturbations caused by excessive folding reactions, acid dissociation was performed in the presence of 0.8 M Na2SO4 at 0°C . Under optimum conditions complete cross-linking of the tetrameric enzyme was achieved in less than 2 minutes. Cross-linking during reconstitution proves the dimer to be the only intermediate of reasso ciation. The dimer → tetramer transition is found to be rate-limiting for both reassociation and reactivation, suggesting the tetramer to be the enzymatically active species. The presence of monomers during reconstitution indicates that tetramer formation is preceded by a fast monomer-dimer equilibrium. The kinetic model describing the experimental data is characterized by an equilibrium constant K = 3 ± 1 · 107 liter · m ol-1, and a second-order rate constant k = 1.4 ± 0.2 · 104 liter · mol-1 · s-1.