Cox R N, Kaldany R R, Brandt P W, Ferren B, Hudson R A, Karlin A
Anal Biochem. 1984 Feb;136(2):476-86. doi: 10.1016/0003-2697(84)90247-1.
A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.
本文描述了一种连续流动技术，其中光亲和标记物、富含乙酰胆碱受体的膜以及各种效应物被快速混合，通过延迟管，再通过一根对其进行照射的管子，最后收集到含有猝灭剂的管子中。混合和光解之间的延迟时间可短至20毫秒。由于流动是连续的，一次运行就能标记数毫升的膜，这便于对光标记的功能效应和位点进行分析。使用该技术，我们发现处于短暂活性状态（通道开放）的受体比处于静息或脱敏状态（通道关闭）的受体更容易被非竞争性抑制剂类似物[3H]喹吖因叠氮化物光标记。该技术对于大分子瞬态构象状态的光标记应具有普遍的实用性。
