Capobianco L, Bisaccia F, Mazzeo M, Palmieri F
Department of Pharmaco-Biology, University of Bari, Italy.
Biochemistry. 1996 Jul 9;35(27):8974-80. doi: 10.1021/bi960258v.
The interaction of sulfhydryl reagents with the oxoglutarate carrier (OGC) of bovine heart mitochondria was investigated in proteoliposomes reconstituted from purified carrier and lipids. Incubation of the proteoliposomes with maleimides or mercurials led to inhibition of the oxoglutarate carrier protein. The inhibition of oxoglutarate transport by mercurials was removed by dithioerythritol (DTE), whereas inhibition by maleimides was not. Preincubation of the proteoliposomes with mercurials protected the carrier protein against inactivation by the fluorescent sulfhydryl reagent N-(1-pyrenyl)maleimide (PM) and decreased the fluorescence associated with the carrier, indicating that mercurials bind to the same cysteine which is modified by PM. The presence of the substrates oxoglutarate and malate increased the binding of PM to the reconstituted carrier as well as the degree of inhibition of the reconstituted transport activity caused by PM, other maleimides, and mercurials. This result is consistent with the assumption that substrate binding causes a change in the tertiary structure of the carrier protein. The primary sequence of the oxoglutarate carrier contains three cysteines (Cys-184, Cys-221, and Cys-224). We provide evidence that PM labels only Cys-184, whereas Cys-221 and Cys-224 are linked by a disulfide bridge.
在由纯化的载体和脂质重构的蛋白脂质体中，研究了巯基试剂与牛心线粒体的草酰戊二酸载体（OGC）之间的相互作用。将蛋白脂质体与马来酰亚胺或汞化合物一起孵育会导致草酰戊二酸载体蛋白受到抑制。汞化合物对草酰戊二酸转运的抑制作用可被二硫苏糖醇（DTE）消除，而马来酰亚胺引起的抑制作用则不能。将蛋白脂质体与汞化合物预孵育可保护载体蛋白不被荧光巯基试剂N-（1-芘基）马来酰亚胺（PM）灭活，并降低与载体相关的荧光，这表明汞化合物与被PM修饰的同一个半胱氨酸结合。底物草酰戊二酸和苹果酸的存在增加了PM与重构载体的结合，以及由PM、其他马来酰亚胺和汞化合物引起的重构转运活性的抑制程度。这一结果与底物结合导致载体蛋白三级结构发生变化的假设一致。草酰戊二酸载体的一级序列包含三个半胱氨酸（Cys-184、Cys-221和Cys-224）。我们提供的证据表明，PM仅标记Cys-184，而Cys-221和Cys-224通过二硫键相连。
