Cherednikova T V, Muronetz V I, Nagradova N K
Biochim Biophys Acta. 1980 Jun 13;613(2):292-308. doi: 10.1016/0005-2744(80)90084-4.
Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CnBr-activated Sepharose via one subunit retain 50% of matrix-bound protein with unaltered specific activity. The solubilized dissociated species are inactive. Two molecules of NAD+ (NADH) firmly bound to the immobilized rat muscle tetramer can prevent the dissociation. Immobilized dimer was demonstrated to bind one molecule of coenzyme with high affinity. Using various combinations of immobilized and soluble rat muscle and yeast dimers, we succeeded in reconstituting tetramers, containing one molecule of NAD+ bound either to a matrix-linked or to a non-covalently bound dimer. In the latter case, the dissociation of the tetramer was completely prevented. This suggests that the binding of a single coenzyme molecule is sufficient to stabilize the interdimeric contacts provided the neighbouring dimer is stabilized independently. Such stabilization is produced by the covalent binding of one of the subunits comprising the dimer to the matrix. The structure of the dimer as a whole becomes resistant to the action of the dissociating agent. The effect appears to be cooperative and similar to that of NAD+ or NADH. The dissociation of the immobilized tetramer is, most likely, the result of conformational changes, affecting the structure of the non-covalently bound dimer. Any factor, capable of preventing these changes, would stabilize the interdimeric contacts. The latter conclusion is substantiated by the effect of specific antibodies, which prevent the dissociation of the immobilized tetramer by forming a complex with the dimer, non-covalently bound to the matrix. The evidence obtained in the present investigation supports the conclusion that the isolated dimer of glyceraldehyde-3-phosphate dehydrogenase represents a relatively independent structural and functional 'unit' of the enzyme. It can be stabilized in a catalytically active form by interactions other than those involved in inter-dimeric contacts in the tetramer. The kinetics of the association of immobilized and soluble dimers have been studied. Association rate constants were determined for homologous (yeast-yeast, rat-rat) and heterologous (yeast-rat, yeast-rabbit) dimer combinations. The binding of one molecule of specific antibody to the immobilized dimer was shown to increase the rate constant of association.
在导致可溶性四聚体甘油醛 - 3 - 磷酸脱氢酶（D - 甘油醛 - 3 - 磷酸：NAD⁺氧化还原酶（磷酸化），EC 1.2.1.12）解离成无活性二聚体的条件下，来自酵母和大鼠骨骼肌的固定化脱辅酶通过一个亚基与溴化氰活化的琼脂糖偶联，保留了50%的基质结合蛋白，且比活性未改变。溶解的解离产物无活性。牢固结合到固定化大鼠肌肉四聚体上的两分子NAD⁺（NADH）可防止解离。已证明固定化二聚体以高亲和力结合一分子辅酶。使用固定化和可溶性大鼠肌肉及酵母二聚体的各种组合，我们成功重构了四聚体，其中一分子NAD⁺要么结合到与基质相连的二聚体上，要么结合到非共价结合的二聚体上。在后一种情况下，四聚体的解离被完全阻止。这表明只要相邻二聚体独立稳定，单个辅酶分子的结合就足以稳定二聚体间的接触。这种稳定作用是由构成二聚体的一个亚基与基质的共价结合产生的。整个二聚体的结构变得对解离剂的作用具有抗性。这种效应似乎是协同的，且与NAD⁺或NADH的效应相似。固定化四聚体的解离很可能是构象变化的结果，这种变化影响了非共价结合二聚体的结构。任何能够阻止这些变化的因素都会稳定二聚体间的接触。后一结论由特异性抗体的作用得到证实，特异性抗体通过与非共价结合到基质上的二聚体形成复合物来阻止固定化四聚体的解离。本研究中获得的证据支持这样的结论，即分离的甘油醛 - 3 - 磷酸脱氢酶二聚体代表了该酶相对独立的结构和功能“单元”。它可以通过四聚体中二聚体间接触以外的相互作用稳定在催化活性形式。已研究了固定化和可溶性二聚体的缔合动力学。测定了同源（酵母 - 酵母、大鼠 - 大鼠）和异源（酵母 - 大鼠、酵母 - 兔）二聚体组合的缔合速率常数。已表明一分子特异性抗体与固定化二聚体的结合会增加缔合速率常数。
