Liang S J, Lin Y Z, Zhou J M, Tsou C L, Wu P Q, Zhou Z K
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, China.
Biochim Biophys Acta. 1990 Apr 19;1038(2):240-6. doi: 10.1016/0167-4838(90)90211-w.
The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.
通过光散射和荧光测量，比较了龙虾肌肉中D-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）（GAPDH）在盐酸胍（GdnHCl）变性过程中的失活情况与其聚集和去折叠状态。该酶在低浓度GdnHCl下首先解离，随后随着变性剂浓度增加形成高度聚集状态，最终在GdnHCl浓度大于2 M时完全去折叠并解离为单体。聚集和最终解离大致与先前报道的荧光变化的两个阶段相对应（谢国藩和邹承鲁（1987年），《生物化学与生物物理学报》911，19 - 24）。速率测量显示失活非常迅速，其程度随着GdnHCl浓度增加而增加。这种在分子解离和去折叠之前发生的初始快速失活阶段与其他酶的结果一致，即在整个酶分子可检测到明显构象变化之前，活性位点就已受到影响。提出了导致酶最终变性并解离为无活性去折叠单体的步骤示意图。
