Marchbanks R M, Israël M
Biochem J. 1972 Oct;129(5):1049-61. doi: 10.1042/bj1291049.
Synaptic vesicles containing radioactive acetylcholine have been isolated from slices of Torpedo electric organ incubated with radioactive choline. The recently synthesized radioactive acetylcholine is preferentially removed from the vesicles by iso-osmotic gel filtration. There is therefore a small compartment of loosely bound recently synthesized acetylcholine within the monodisperse vesicle fraction. The specific radioactivity of this compartment correlates most closely with the ;free' acetylcholine of electric organ that is lost when the tissue is homogenized. Membrane-associated vesicles did not contain any particular enrichment of this compartment. On standing at 6 degrees C the loosely bound compartment stabilizes so that it survives iso-osmotic filtration. A study of this phenomenon revealed that it was proportional to the extent of the loss of tightly bound acetylcholine from the vesicles. Incubation with Ca(2+), at pH5.5, or partial hypo-osmotic shock, caused losses of tightly bound acetylcholine and proportional increases in the stabilization of loosely bound acetylcholine of vesicles. Incubation at 20 degrees C caused less loss of tightly bound, and less stabilization of loosely bound, acetylcholine. A theoretical treatment of these exchanges also shows that the random factors promoting loss of tightly bound acetylcholine are statistically correlated with those which cause stabilization of loosely bound acetylcholine. The reciprocal relationship between the exchanges is inconsistent with there being two distinct populations of vesicles, one containing recently synthesized, loosely bound acetylcholine and the other containing tightly bound acetylcholine. It is proposed that all the vesicles contain a core of tightly bound acetylcholine and a surface layer of loosely bound acetylcholine. The origin of the extravesicular acetylcholine and also of the acetylcholine released on stimulation is discussed in the light of these results.
已从用放射性胆碱孵育的电鳐电器官切片中分离出含有放射性乙酰胆碱的突触小泡。通过等渗凝胶过滤，优先从这些小泡中去除最近合成的放射性乙酰胆碱。因此，在单分散小泡部分中存在一小部分结合松散的最近合成的乙酰胆碱。该部分的比放射性与电鳐器官中“游离”乙酰胆碱的比放射性最密切相关，当组织匀浆时，该“游离”乙酰胆碱会丢失。与膜相关的小泡并不含有该部分的任何特殊富集。在6℃下放置时，结合松散的部分会稳定下来，从而在等渗过滤后仍能留存。对这一现象的研究表明，它与小泡中紧密结合的乙酰胆碱的丢失程度成正比。在pH5.5下用Ca(2+)孵育或部分低渗休克，会导致紧密结合的乙酰胆碱丢失，并使小泡中结合松散的乙酰胆碱的稳定性成比例增加。在20℃下孵育导致紧密结合的乙酰胆碱丢失较少，结合松散的乙酰胆碱的稳定性增加也较少。对这些交换的理论处理还表明，促进紧密结合的乙酰胆碱丢失的随机因素与导致结合松散的乙酰胆碱稳定的因素在统计学上相关。这些交换之间的相互关系与存在两种不同的小泡群体不一致，一种含有最近合成的、结合松散的乙酰胆碱，另一种含有紧密结合的乙酰胆碱。有人提出，所有的小泡都含有一个紧密结合的乙酰胆碱核心和一个结合松散的乙酰胆碱表面层。根据这些结果，讨论了细胞外乙酰胆碱以及刺激时释放的乙酰胆碱的来源。
