Klingenberg M, Mayer I, Appel M
Biochemistry. 1985 Jul 2;24(14):3650-9. doi: 10.1021/bi00335a038.
The binding of the 3'-[1,5-(dimethylamino)naphthoyl] (DAN) derivatives of AMP, ADP, and ATP to the solubilized ADP/ATP carrier is studied, evaluating primarily the fluorescence enhancement and 3H-labeled compound binding. DAN nucleotides also fluoresce when adsorbed to Triton X-100 micelles that are used for solubilization of the carrier. The partition of DAN-AMP between water and Triton X-100 micelles is measured, and it is shown to be shifted toward a higher content in Triton micelles with increasing salt concentration. In order to maintain a low level of fluorescence, the Triton content is decreased. The fraction of DAN nucleotide fluorescence due to carrier binding is determined by the suppression with bongkrekate (BKA). In contrast to the membrane-bound carrier, the solubilized preparation shows an increase of total BKA-sensitive fluorescence by 30-60% upon addition of ATP or ADP. In the solubilized atractylate-protein complex, the ADP-stimulated fluorescence amounts even to 80%. The suppression of fluorescence by BKA is independent of the presence of ADP or ATP, while that by carboxyatractylate (CAT) depends on ADP or ATP. The quantitation with [3H]BKA and [3H]CAT of these ligand interactions with DAN-AMP fluorescence shows that DAN-AMP fluorescence reflects the "m"-state carrier population and its redistribution under the influence of ADP or ATP. Thus, besides the "c"/"m" distribution, the kinetics of the c to m transition in the solubilized carrier also can be determined. The m share is increased to 80% when SO4, Pi, or pyrophosphate is present during solubilization. The rate of the ADP- or ATP-stimulated transition to the m state is markedly dependent on pH and on the presence of various anions, whereas the extent is little varied. The affinity decreases 4-fold going from DAN-AMP to DAN-ADP and to DAN-ATP (KD = 0.9, 1.6, and 3.2 microM). Comparison with physical binding of [3H]DAN nucleotides shows that the fluorescence yield of bound DAN-AMP is about 1.4 times higher than that of bound DAN-ATP. DAN substitution causes more than a 100-fold affinity increase for AMP and a 50-fold increase for ADP or ATP, probably because of interaction of the DAN group with a hydrophobic niche. A less specific, low-affinity displacement of DAN nucleotides by GDP, ADP, GTP and ATP (Ki = 1-2 mM) probably reflects primarily the ionic interactions at the binding center.
研究了AMP、ADP和ATP的3'-[1,5-(二甲基氨基)萘甲酰基]（DAN）衍生物与溶解的ADP/ATP载体的结合，主要评估荧光增强和3H标记化合物的结合情况。当DAN核苷酸吸附到用于溶解载体的Triton X-100胶束上时也会发出荧光。测量了DAN-AMP在水和Triton X-100胶束之间的分配情况，结果表明随着盐浓度的增加，它在Triton胶束中的含量会向更高水平偏移。为了保持低荧光水平，降低了Triton的含量。通过用邦克雷酸（BKA）抑制来确定由于载体结合导致的DAN核苷酸荧光分数。与膜结合载体不同，溶解制剂在加入ATP或ADP后，总BKA敏感荧光增加30 - 60%。在溶解的苍术苷 - 蛋白质复合物中，ADP刺激的荧光甚至达到80%。BKA对荧光的抑制与ADP或ATP的存在无关，而羧基苍术苷（CAT）的抑制则取决于ADP或ATP。用[3H]BKA和[3H]CAT对这些配体与DAN-AMP荧光的相互作用进行定量分析表明，DAN-AMP荧光反映了“m”态载体群体及其在ADP或ATP影响下的重新分布。因此，除了“c”/“m”分布外，还可以确定溶解载体中c到m转变的动力学。当在溶解过程中存在SO4、Pi或焦磷酸时，“m”态的比例增加到80%。ADP或ATP刺激向“m”态转变的速率明显取决于pH值和各种阴离子的存在，而转变程度变化不大。从DAN-AMP到DAN-ADP再到DAN-ATP，亲和力降低4倍（KD = 0.9、1.6和3.2 microM）。与[3H]DAN核苷酸的物理结合比较表明，结合的DAN-AMP的荧光产率比结合的DAN-ATP高约1.4倍。DAN取代使对AMP的亲和力增加超过100倍，对ADP或ATP的亲和力增加50倍，这可能是由于DAN基团与疏水位点的相互作用。GDP、ADP、GTP和ATP对DAN核苷酸的非特异性、低亲和力置换（Ki = 1 - 2 mM）可能主要反映了结合中心的离子相互作用。
