Stappen R, Dierks T, Bröer A, Krämer R
Institut für Biotechnologie 1, Forschungszentrum Jülich, Federal Republic of Germany.
Eur J Biochem. 1992 Nov 15;210(1):269-77. doi: 10.1111/j.1432-1033.1992.tb17417.x.
Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or glutamate, 50% substrate protection being observed at half-saturation of the external binding site. The bifunctional reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) also modified the same cysteine and, in addition, an active-site lysine identified previously [Dierks, T., Stappen, R., Salentin, A. & Krämer, R. (1992) Biochim. Biophys. Acta 1103, 13-24]. The proximity of the cysteine [Cys(a)] and the lysine residue was confirmed by a mutual exclusion of the respective reagents when added consecutively. By using a variety of reagents a further cysteine [Cys(b)] and probably a histidine residue could be discriminated from Cys(a) and the lysine. The applied reagents were classified according to functional and structural criteria. Class A reagents, like Nbd-Cl, modified the active-site Cys(a) thereby inhibiting the antiport function. Class B reagents, like HgCl2, reacted with both Cys(a) and Cys(b) leading to a conversion of the carrier from antiport to uniport function [Dierks, T., Salentin, A., Heberger, C. & Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268-280]. DIDS at relatively high concentration (60 microM) also acted as a uniport inducer. Class C reagents finally, like pyridoxal phosphate or diethyl pyrocarbonate, modified the active-site lysine or histidine, respectively, and blocked antiport and uniport activity. By testing the accessibility of the mentioned residues to the various reagents, when applied in different order, topological relationships could be elaborated indicating the location of these amino acids with respect to the exofacial active site of the carrier protein.
用7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂（Nbd-Cl）处理来自线粒体的重组天冬氨酸/谷氨酸载体，导致载体功能完全失活。抑制作用可归因于活性位点中单个半胱氨酸的化学修饰。在天冬氨酸或谷氨酸存在下，该残基受到特异性保护，在外源性结合位点半饱和时观察到50%的底物保护。双功能试剂4,4'-二异硫氰基芪-2,2'-二磺酸盐（DIDS）也修饰了同一个半胱氨酸，此外，还修饰了先前鉴定的一个活性位点赖氨酸[迪克斯，T.，施塔彭，R.，萨伦廷，A. & 克雷默，R.（1992年）《生物化学与生物物理学学报》1103，13 - 24]。当连续添加时，各自试剂相互排斥，证实了半胱氨酸[Cys(a)]和赖氨酸残基的接近性。通过使用多种试剂，可以将另一个半胱氨酸[Cys(b)]以及可能的一个组氨酸残基与Cys(a)和赖氨酸区分开来。根据功能和结构标准对所应用的试剂进行了分类。A类试剂，如Nbd-Cl，修饰活性位点的Cys(a)，从而抑制反向转运功能。B类试剂，如HgCl2，与Cys(a)和Cys(b)都发生反应，导致载体从反向转运功能转变为单向转运功能[迪克斯，T.，萨伦廷，A.，赫贝格尔，C. & 克雷默，R.（1990年）《生物化学与生物物理学学报》1028，268 - 280]。相对高浓度（60 microM）的DIDS也作为单向转运诱导剂起作用。最后，C类试剂，如磷酸吡哆醛或焦碳酸二乙酯，分别修饰活性位点的赖氨酸或组氨酸，并阻断反向转运和单向转运活性。通过测试上述残基对各种试剂的可及性，当以不同顺序应用时，可以阐明拓扑关系，表明这些氨基酸相对于载体蛋白外表面活性位点的位置。
