Tuena de Gómez-Puyou M, Sandoval F, Gómez-Puyou A
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D. F., México.
J Biol Chem. 1995 Jul 14;270(28):16820-5. doi: 10.1074/jbc.270.28.16820.
Soluble F1 from heart mitochondria incubated in mixtures that have Mg2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., García, J. J., and Gómez-Puyou, A. (1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration experiments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PPi in F1 showed that hydrolysis of synthesized PPi at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PPi but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F1 that had been depleted of adenine nucleotides. Hence, dilution-induced release of PPi was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium Pi and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F1.
在含有镁离子、无机磷酸盐和二甲基亚砜（40%（v/v））的混合物中孵育的心脏线粒体可溶性F1催化ATP和焦磷酸的自发合成（图埃纳·德·戈麦斯 - 皮尤、M.、加西亚、J. J.和戈麦斯 - 皮尤、A.（1993年）《生物化学》32卷，2213 - 2218页）。通过过滤技术确定，合成的ATP和焦磷酸与酶结合，尽管其对焦磷酸的亲和力低于对ATP的亲和力。在二甲基亚砜混合物中形成ATP和焦磷酸后，用水性缓冲液稀释至二甲基亚砜浓度为6.0%会导致焦磷酸分配到介质中。过滤实验以及合成的焦磷酸对可溶性无机焦磷酸酶的可及性都证明了这一点。稀释诱导的焦磷酸释放发生在不到15秒内。在产生焦磷酸释放的条件下，未观察到ATP释放；相反，ATP发生了水解。关于砷酸盐对F1中ATP和PPi合成及水解的影响的研究表明，合成的PPi在其合成位点的水解比ATP慢。因此，探讨了水解速率的差异是否是稀释诱导PPi而非ATP释放的原因。在可溶性F1与其抑制蛋白形成的复合物制剂中检测了ATP和焦磷酸的合成及水解；该复合物的ATP酶活性比游离F1低约100倍。在含有二甲基亚砜的混合物中，该复合物以几乎相同的速率合成ATP和焦磷酸；稀释后，两种化合物的水解也以相似的速率发生，但只有焦磷酸被释放。在已耗尽腺嘌呤核苷酸的F1中也观察到了相同的现象。因此，稀释诱导的PPi释放与酶的整体催化特性或其腺嘌呤核苷酸含量无关。由于ATP的合成是以腺嘌呤核苷酸耗尽后剩余的ADP为代价发生的，ATP未能释放可能是因为F1对合成的ATP表现出高亲和力。然而，结果表明，一个从介质中的Pi开始并以介质中的焦磷酸结束的完整催化循环可以在可溶性线粒体F1中重现。
