Rasched I R, Bohn A, Sund H
Eur J Biochem. 1977 Apr 1;74(2):365-77. doi: 10.1111/j.1432-1033.1977.tb11401.x.
Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with mono-functional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.
用己二酸二甲酯对牛肝谷氨酸脱氢酶单体（由六条分子量均为56000的多肽链组成）进行交联，随后通过十二烷基硫酸钠电泳分析，结果显示主要为三聚体形式（分子量168000）。用其他链长的二甲酯处理，产生的三聚体形式显著减少，这表明被交联的氨基间距约为0.85纳米。将掺入的[¹⁴C]己二酸二甲酯的摩尔量与修饰氨基的数量（用三硝基苯磺酸测定）进行比较，结果表明，虽然34个氨基中有8 - 9个发生了反应，但其中只有2 - 3个参与了交联。与二甲酯反应会使酶失活。活性的丧失部分与三聚体形式的交联相伴发生，而不仅仅是由于必需赖氨酸残基的修饰。这一观点得到以下事实的支持：虽然更多的赖氨酸残基与单功能甲基酯反应，但活性丧失程度降低。对放射性标记的三聚体形式的纯化胰凝乳蛋白酶和胰蛋白酶肽段进行序列分析。鉴定出六个含有总标记75%的肽段：一个涉及氨基末端残基丙氨酸 - 1，其他的涉及赖氨酸 - 105、赖氨酸 - 154、赖氨酸 - 269、赖氨酸 - 358和赖氨酸 - 399。对每个肽段/摩尔赖氨酸的比放射性进行定量分析得出结论，只有赖氨酸 - 105、赖氨酸 - 154、赖氨酸 - 269和赖氨酸 - 358参与交联，赖氨酸 - 269和赖氨酸 - 358分别位于同源接触区域，赖氨酸 - 105在酶的异源接触区域与赖氨酸 - 154交联。讨论了谷氨酸脱氢酶四级结构中三聚体形式的平面排列模型。它包括多肽链之间的同源和异源接触区域。
