Hüther F J, Kadenbach B
FEBS Lett. 1986 Oct 20;207(1):89-94. doi: 10.1016/0014-5793(86)80018-7.
Bovine heart cytochrome-c oxidase was reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured by the polarographic and photometric method under uncoupled conditions in the presence of various polyvalent anions. In order to distinguish between specific and unspecific ionic effects of ATP, the photolabelling reagent 8-azido-ATP was applied. Covalently bound ATP at the enzyme complex caused the same increase of Km for cytochrome c as free ATP, if measured by the photometric assay. The increase of Km by photolabelling with 8-azido-ATP was completely prevented by ATP, but not by ADP. The data indicate the occurrence of a specific binding site for ATP at the cytosolic side of cytochrome-c oxidase, which, after binding of ATP, changes the kinetics of cytochrome c oxidation.
牛心细胞色素c氧化酶被重组到脂质体中，在未偶联的条件下，于各种多价阴离子存在时，通过极谱法和光度法测量细胞色素c氧化的动力学。为了区分ATP的特异性和非特异性离子效应，应用了光标记试剂8-叠氮基-ATP。如果通过光度测定法测量，酶复合物上共价结合的ATP导致细胞色素c的Km增加与游离ATP相同。用8-叠氮基-ATP进行光标记导致的Km增加被ATP完全阻止，但不被ADP阻止。数据表明在细胞色素c氧化酶的胞质侧存在一个ATP特异性结合位点，该位点在结合ATP后会改变细胞色素c氧化的动力学。
