Block M R, Vignais P V
Biochim Biophys Acta. 1984 Nov 26;767(2):369-76. doi: 10.1016/0005-2728(84)90207-x.
The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid. The nucleotides tested fell into two classes depending on the shape of the binding curve. Curvilinear Scatchard plots were obtained for the binding of ADP, ATP, adenosine 5'-triphospho-gamma-1-(5-sulfonic acid)naphthylamidate [gamma-AmNS)ATP) and adenylyl (beta,gamma)-methylenediphosphate (p[CH2]ppA); on the other hand, rectilinear Scatchard plots were obtained in the case of naphthoyl-ADP (N-ADP) and 8-bromo ADP (8Br-ADP) binding. The total number of binding sites for N-ADP and 8Br-ADP could be extrapolated with good accuracy to 1.3-1.5 nmol/mg protein; this value corresponds to the number of carboxyatractyloside-binding sites in heart mitochondria (Block, M.R., Pougeois, R. and Vignais, P.V. (1980) FEBS Lett. 117, 335-340). On the other hand, because of the curvilinearity of the Scatchard plots for the binding of ADP, ATP, (gamma-AmNS)ATP and p[CH2]ppA, the total number of binding sites for these nucleotides could only be approximated to a value higher than 1 nmol/mg protein, the exact value being probably equal to that found for N-ADP and 8Br-ADP binding, i.e. 1.3-1.5 nmol/mg protein. Curvilinearity of Scatchard plots was discussed in terms of negative interactions between nucleotide-binding sites located on the same face of the adenine nucleotide carrier. A possible relationship between the features of the binding plots and the transportable nature of the nucleotide is discussed. Contrary to the enhancing effect of bongkrekic acid on [14C]ADP uptake observed essentially in nucleotide-depleted heart mitochondria (Klingenberg, M., Appel, M., Babel, W. and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654), binding of bongkrekic acid to nondepleted heart mitochondria was found to partially displace previously bound [14C]ADP. These opposite effects of bongkrekic acid may be explained by assuming that bongkrekic acid is able to abolish negative cooperativity between external (cytosolic) ADP-binding sites.
利用两种特异性抑制剂——羧基苍术苷和邦克酸，研究了多种ADP或ATP类似物与线粒体及外翻亚线粒体颗粒中腺嘌呤核苷酸载体的结合参数。根据结合曲线的形状，所测试的核苷酸分为两类。ADP、ATP、腺苷5'-三磷酸-γ-1-(5-磺酸)萘酰胺[γ-AmNS)ATP]和腺苷酰(β,γ)-亚甲基二磷酸(p[CH2]ppA)的结合得到的是曲线型Scatchard图；另一方面，萘甲酰基-ADP (N-ADP)和8-溴ADP (8Br-ADP)的结合得到的是直线型Scatchard图。N-ADP和8Br-ADP的结合位点总数可准确外推至1.3 - 1.5 nmol/mg蛋白质；该值与心脏线粒体中羧基苍术苷结合位点的数量相对应(Block, M.R., Pougeois, R.和Vignais, P.V. (1980) FEBS Lett. 117, 335 - 340)。另一方面，由于ADP、ATP、(γ-AmNS)ATP和p[CH2]ppA结合的Scatchard图呈曲线状，这些核苷酸的结合位点总数只能近似为高于1 nmol/mg蛋白质的值，确切值可能与N-ADP和8Br-ADP结合时的值相等，即1.3 - 1.5 nmol/mg蛋白质。根据位于腺嘌呤核苷酸载体同一面上的核苷酸结合位点之间的负相互作用，对Scatchard图的曲线性进行了讨论。讨论了结合图的特征与核苷酸可转运性质之间的可能关系。与主要在核苷酸耗尽的心脏线粒体中观察到的邦克酸对[14C]ADP摄取的增强作用相反(Klingenberg, M., Appel, M., Babel, W.和Aquila, H. (1983) Eur. J. Biochem. 131, 647 - 654)，发现邦克酸与未耗尽的心脏线粒体的结合会部分取代先前结合的[14C]ADP。邦克酸的这些相反作用可以通过假设邦克酸能够消除外部(胞质)ADP结合位点之间的负协同作用来解释。
