Czarnecki J J, Abbott M S, Selman B R
Eur J Biochem. 1983 Oct 17;136(1):19-24. doi: 10.1111/j.1432-1033.1983.tb07699.x.
The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.
光亲和类似物2-叠氮基-ADP（2-叠氮腺苷5'-二磷酸）被用作菠菜叶绿体ATP合酶的探针。该类似物可作为光合磷酸化的底物。多项观察结果表明，2-叠氮基-ADP和ADP结合到同一类紧密核苷酸结合位点上：（a）2-叠氮基-ADP竞争性抑制ADP的紧密结合（Ki = 1.4 microM）；（b）使类似物紧密结合达到最大结合量50%时的浓度，即K0.5（1 microM）与ADP的K0.5（2 microM）相似；（c）核苷酸紧密结合需要预先使膜产生能量，且重新产生能量可使其完全逆转；（d）ADP可完全阻止2-叠氮基-[β-32P]ADP的紧密结合；（e）该类似物在微摩尔浓度下可抑制光触发的ATP酶活性。对含有紧密结合的2-叠氮基-[β-32P]ADP的洗涤类囊体膜进行紫外线照射，导致标记物共价掺入膜中。对标记膜进行变性聚丙烯酰胺凝胶电泳表明，偶联因子一复合物的β亚基是类囊体膜中唯一被标记的多肽。这些结果确定了偶联因子的β亚基是类囊体膜上紧密结合ADP的位置。
