Fedan J S, Hogaboom G K, O'Donnell J P, Jeng S J, Guillory R J
Eur J Pharmacol. 1985 Jan 15;108(1):49-61. doi: 10.1016/0014-2999(85)90282-1.
Following its photolysis in the presence of the isolated guinea-pig vas deferens, the ATP photoaffinity label ANAPP3 produces a specific antagonism of adenine nucleotide-induced contractile responses which are mediated by P2-purinergic receptors. To characterize the site of covalent photoincorporation of ANAPP3, intact vasa deferentia were treated with [3H]ANAPP3 and samples of homogenate, cytosol and a crude membrane fraction were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Photolysis of [3H]ANAPP3 (10(-5) M; 3.0 mu Ci/ml) resulted in the incorporation of radioactivity into cellular components with apparent molecular weights of 54-66 and 43-57 kilodaltons. The photoincorporation of [3H]ANAPP3 was associated with the crude membrane fraction and not the cytosol, was reduced in the presence of ATP in an ATP-concentration-dependent manner, was lessened following pretreatment of the tissues with photolyzed nonradiolabeled ANAPP3, and was unaffected by the nucleoside transport inhibitor, dipyridamole. In tension studies on the same tissues the presence of ATP resulted in a concentration-dependent reduction in the initial contractile response to [3H]ANAPP3 the response to 3H was antagonized in tissues which had been pretreated with nonradiolabeled ANAPP3, and dipyridamole had no effect on the contractile response to [3H]ANAPP3. According to several criteria these findings indicate that the antagonism by photolyzed ANAPP3 of adenine nucleotide-induced responses is a direct result of the covalent insertion at or near the recognition site of cell-surface P2-purinergic receptors.
在分离的豚鼠输精管存在的情况下，ATP光亲和标记物ANAPP3发生光解后，会对由P2嘌呤能受体介导的腺嘌呤核苷酸诱导的收缩反应产生特异性拮抗作用。为了确定ANAPP3共价光掺入的位点，用[3H]ANAPP3处理完整的输精管，然后使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析匀浆、胞质溶胶和粗膜部分的样品。[3H]ANAPP3（10^(-5) M；3.0 μCi/ml）的光解导致放射性掺入细胞成分，其表观分子量为54 - 66千道尔顿和43 - 57千道尔顿。[3H]ANAPP3的光掺入与粗膜部分有关，而不是胞质溶胶，在ATP存在下以ATP浓度依赖的方式减少，在用光解的非放射性标记ANAPP3预处理组织后减少，并且不受核苷转运抑制剂双嘧达莫的影响。在对相同组织的张力研究中，ATP的存在导致对[3H]ANAPP3的初始收缩反应呈浓度依赖性降低，在用非放射性标记ANAPP3预处理的组织中对3H的反应受到拮抗，并且双嘧达莫对[3H]ANAPP3的收缩反应没有影响。根据几个标准，这些发现表明光解的ANAPP3对腺嘌呤核苷酸诱导反应的拮抗作用是细胞表面P2嘌呤能受体识别位点处或附近共价插入的直接结果。
