Klein G, Satre M, Dianoux A C, Vignais P V
Biochemistry. 1981 Mar 3;20(5):1339-44. doi: 10.1021/bi00508a046.
The natural mitochondrial ATPase inhibitor (IF1) was modified with a radioactivity labeled heterobifunctional and photosensitive reagent, methyl 4-azido(14C)benzimidate ((14C)MABI). Titration experiments of IF1 by (14C)MABI and tryptic maps of (14C)MABI-IF1 indicated that specific lysine residues in IF1 are preferentially labeled by (14C)MABI. Under appropriate conditions of labeling (1 to 2 lysine residues modified per IF1), MABI-IF1 exhibited the same inhibitory potency as native IF1 on the hydrolytic activity of the coupling factor 1 of mitochondrial ATPase (F1). The same conditions were required for inhibition of F1 by MABI-IF1 and IF1 (slightly acidic pH and presence of ATP and MgCl2). In photolabeling experiments, (14C)MABI-IF1 was used to investigate the localization of IF1 binding sites on F1. Upon photoirradiation, MABI-IF1 bound selectively to the beta subunit of soluble or membrane-bound F1. Adenylyl imidodiphosphate and quercetin, two compounds which partially mimic the inhibitory effect of IF1 on ATPase activity of F1, markedly prevented the binding of (14C)MABI-IF1 to F1; on the other hand, aurovertin, a specific ligand of the beta subunit of F1, did not affect the interaction between (14C)MABI-IF1 and F1. In the absence of light, (14C)MABI-IF1 was used as a reversible radiolabeled ligand with respect to membrane bound F1 to investigate F1-IF1 interactions to inside-out submitochondrial particles as a function of the energy state of the particles. Oxidation of NADH by submitochondrial particles resulted in a decrease of bound (14C)MABI-IF1; the effect was counteracted by antimycin. The data suggested that added (14C)MABI-IF1 is capable of exchanging with IF1 bound to F1 in submitochondrial particles and that the rate and extent of (14C)MABI-IF1 release are triggered by the proton-motive force developed by the particles.
天然线粒体ATP酶抑制剂（IF1）用放射性标记的异双功能光敏试剂4-叠氮基（14C）苯甲酸甲酯（（14C）MABI）进行修饰。用（14C）MABI对IF1进行滴定实验以及（14C）MABI-IF1的胰蛋白酶图谱表明，IF1中的特定赖氨酸残基优先被（14C）MABI标记。在适当的标记条件下（每个IF1修饰1至2个赖氨酸残基），MABI-IF1对线粒体ATP酶偶联因子1（F1）的水解活性表现出与天然IF1相同的抑制效力。MABI-IF1和IF1抑制F1需要相同的条件（略酸性pH以及ATP和MgCl2的存在）。在光标记实验中，（14C）MABI-IF1用于研究IF1在F1上的结合位点的定位。光照后，MABI-IF1选择性地结合到可溶性或膜结合F1的β亚基上。腺苷酰亚胺二磷酸和槲皮素这两种部分模拟IF1对F1的ATP酶活性抑制作用的化合物，显著阻止了（14C）MABI-IF1与F1的结合；另一方面，F1的β亚基的特异性配体金轮菌素不影响（14C）MABI-IF1与F1之间的相互作用。在无光条件下，（14C）MABI-IF1用作相对于膜结合F1的可逆放射性标记配体，以研究F1-IF1与内翻线粒体亚颗粒的相互作用与颗粒能量状态的关系。线粒体亚颗粒对NADH的氧化导致结合的（14C）MABI-IF1减少；抗霉素可抵消该效应。数据表明，添加的（14C）MABI-IF1能够与结合在线粒体亚颗粒中F1上的IF1进行交换，并且（14C）MABI-IF1释放的速率和程度由颗粒产生的质子动力触发。
