Abbott M S, Czarnecki J J, Selman B R
J Biol Chem. 1984 Oct 10;259(19):12271-8.
The photoaffinity analog 2-azido-ADP has been used to investigate the high-affinity binding site(s) for ATP on the chloroplast thylakoid membrane. Photophosphorylation of 2-azido-ADP results in the rapid formation of 2-azido-ATP, which remains tightly bound to the membranes after extensive washing. The kinetic parameters of the tight binding of ATP and of 2-azido-ATP are similar (apparent Km = 1-2 microM; maximum extent = 0.2-0.4 nmol/mg of chlorophyll). Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[gamma-32P]ATP induces covalent incorporation of the label exclusively into the beta subunit of the chloroplast coupling factor one. Previous results have shown that the tight binding site for ADP is also located on the beta subunit of the ATP synthase (Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24). To further characterize the tight binding sites for ADP and ATP, the membrane-bound coupling factor has been covalently modified with either tightly bound 2-azido-[gamma-32P]ATP or tightly bound 2-azido-[beta-32P]ADP. The photolabeled beta subunits have been isolated and subjected to partial proteolytic digestion and SDS-gel electrophoresis. The results of these experiments demonstrate that the tight binding sites for ADP and ATP are located on identical portions of beta subunit polypeptide.
光亲和类似物2-叠氮基-ADP已被用于研究叶绿体类囊体膜上ATP的高亲和力结合位点。2-叠氮基-ADP的光磷酸化导致2-叠氮基-ATP的快速形成，经过大量洗涤后，它仍紧密结合在膜上。ATP和2-叠氮基-ATP紧密结合的动力学参数相似（表观Km = 1-2微摩尔；最大结合量 = 0.2-0.4纳摩尔/毫克叶绿素）。对含有紧密结合的2-叠氮基-[γ-32P]ATP的洗涤后的类囊体膜进行紫外线照射，可诱导标记物仅共价掺入叶绿体偶联因子1的β亚基中。先前的结果表明，ADP的紧密结合位点也位于ATP合酶的β亚基上（Czarnecki, J. J., Abbott, M. S., and Selman, B. R. (1983) Eur. J. Biochem. 136, 19-24）。为了进一步表征ADP和ATP的紧密结合位点，已用紧密结合的2-叠氮基-[γ-32P]ATP或紧密结合的2-叠氮基-[β-32P]ADP对膜结合的偶联因子进行了共价修饰。已分离出光标记的β亚基，并对其进行部分蛋白酶解消化和SDS-凝胶电泳。这些实验结果表明，ADP和ATP的紧密结合位点位于β亚基多肽的相同部分。
