van Dongen M B, Berden J A
Biochim Biophys Acta. 1986 Jun 10;850(1):121-30. doi: 10.1016/0005-2728(86)90016-2.
The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.
光反应性核苷酸[2-³H]8-叠氮基-ATP和[2-³H]8-叠氮基-ADP可用于标记分离的线粒体F1-ATP酶上的核苷酸结合位点,即使F1中紧密结合的核苷酸已耗尽,每摩尔F1最多可结合4摩尔核苷酸。在300-1000微摩尔的光标记浓度下,α和β亚基上均能发现标记,其典型比例为1:3,与结合的总量无关。在这些条件下,两个核苷酸的共价结合是完全失活所必需的(瓦根沃德,R.J.,范德克兰,I.和肯普,A.(1977年)《生物化学与生物物理学报》460,17-24)。在较低浓度的[2-³H]8-叠氮基-ATP(20微摩尔)下,发现每摩尔F1仅需1摩尔核苷酸的共价结合即可完全失活,这表明ATP水解机制中存在催化位点协同作用。在这些条件下,放射性仅在β亚基上发现,这表明催化位点位于β亚基上,而第二个位点位于α/β界面上。发现六个核苷酸结合位点中有四个是可交换的,并且可以用8-叠氮基-AT(D)P标记,即两个催化位点和两个非催化位点。