Klingenberg M, Appel M, Babel W, Aquila H
Eur J Biochem. 1983 Apr 5;131(3):647-54. doi: 10.1111/j.1432-1033.1983.tb07312.x.
The binding of bongkrekate to mitochondrial membrane was investigated using [3H]bongkrekate. These measurements were designed to examine the previously derived reorienting site mechanism which implies that bongkrekate binds to the single carrier site only from the inner face of the mitochondrial membrane. The binding studies confirm pH-dependent accumulation of [3H]bongkrekate inside the mitochondria which superimposes on to binding of carrier sites. By breaking the membrane with Lubrol or sonication, binding to the carrier sites can be titrated and Kd approximately equal to 5 X 10(-8) M is determined. ADP or ATP increases the amount of binding but does not change the Kd. Reciprocally bongkrekate increases ADP binding in those sections of a titration curve where bongkrekate binding is increased by ADP. [35S]Atractylate is displaced by [3H]bongkrekate at a 1:1 molar ratio. This displacement is dependent on ADP concentration with the Km = 0.5 X 10(-6) M. The earlier described isomer, isobongkrekate, also binds specifically to the carrier sites. From competition with bongkrekate a ratio KisoBKAd/KBKAd = 0.10 is determined. [35S]Carboxyatractylate displaces most of [3H]isobongkrekate but only little of [3H]bongkrekate. The rate of displacement is more than 10-times faster for isobongkrekate than for bongkrekate. The displacement is dependent on ADP with a Km = 5 X 10(-6) M. All these results are fully consistent with the single site reorienting mechanism. In no instant do bongkrekate and atractylate as well as ADP or ATP bind simultaneously to the carrier.
使用[³H]邦克雷酸研究了邦克雷酸与线粒体膜的结合。这些测量旨在检验先前推导的重定向位点机制，该机制表明邦克雷酸仅从线粒体内膜表面结合到单一载体位点。结合研究证实了[³H]邦克雷酸在线粒体内的pH依赖性积累，这种积累叠加在载体位点的结合上。通过用卢勃罗尔（Lubrol）或超声破碎膜，可以滴定与载体位点的结合，并确定解离常数（Kd）约等于5×10⁻⁸ M。二磷酸腺苷（ADP）或三磷酸腺苷（ATP）增加结合量，但不改变Kd。相应地，在滴定曲线中，当ADP增加邦克雷酸结合量的那些部分，邦克雷酸会增加ADP的结合。[³⁵S]苍术苷（Atractylate）以1:1的摩尔比被[³H]邦克雷酸取代。这种取代依赖于ADP浓度，米氏常数（Km）= 0.5×10⁻⁶ M。先前描述的异构体异邦克雷酸也特异性地结合到载体位点。通过与邦克雷酸的竞争，确定了一个比例KisoBKAd/KBKAd = 0.10。[³⁵S]羧基苍术苷取代了大部分[³H]异邦克雷酸，但仅取代了少量[³H]邦克雷酸。异邦克雷酸的取代速率比邦克雷酸快10倍以上。这种取代依赖于ADP，Km = 5×10⁻⁶ M。所有这些结果与单一位点重定向机制完全一致。邦克雷酸和苍术苷以及ADP或ATP在任何时候都不会同时结合到载体上。
