Brockman S K, Younkin L H, Younkin S G
J Neurosci. 1984 Jan;4(1):131-40. doi: 10.1523/JNEUROSCI.04-01-00131.1984.
We have investigated the effect of electromechanical activity on the molecular forms of acetylcholinesterase (AChE) in cultured embryonic rat myotubes. Both globular and asymmetric forms of AChE are present on the 5th day of culture when myotubes are just beginning to fibrillate. Between days 5 and 8, the 4 S (G1), 10 S (G4), and 16 S (A12) forms increase dramatically, and appreciable 12.5 S (A8) AChE appears. When fibrillation is prevented by adding tetrodotoxin on day 4, the increases in the A12 and A8 forms are prevented, and the increases in the G4 and G1 forms are significantly impaired. At 8 days, fibrillating myotubes have 19 times more A12 AChE and over 4 times more G1 and G4 enzyme than do nonfibrillating myotubes. The effect of tetrodotoxin is reversible. When tetrodotoxin is removed at 7 days, fibrillation resumes promptly, and globular and asymmetric forms recover. Light microscopic examination of fibrillating and nonfibrillating myotubes showed that tetrodotoxin does not affect the gross morphological development of the myotubes. Titration of AChE-active sites with O-ethyl-S2-diisopropyl methyl-phosphonothionate demonstrated that the increase in AChE activity associated with fibrillation is due to an increase in the number of AChE molecules present and not to an increase in the rate at which individual AChE molecules turn over acetylcholine. To evaluate AChE metabolism in fibrillating and nonfibrillating myotubes, we examined the enzyme after inactivating it with paraoxon. Paraoxon readily penetrates cells and diethylphosphorylates a serine in the active site of AChE, thereby inactivating it. The diethylphosphorylated enzyme is stable, but it can be reactivated rapidly and quantitatively with pyridine-2-aldoxime methiodide (2-PAM). After inactivating AChE with paraoxon, we simultaneously evaluated synthesis (by following the newly synthesized active AChE) and turnover (by following the 2-PAM-reactivatable AChE). Our results show that globular and asymmetric forms of AChE are both synthesized more rapidly in fibrillating than in nonfibrillating myotubes.
我们研究了机电活动对培养的胚胎大鼠肌管中乙酰胆碱酯酶（AChE）分子形式的影响。在培养的第5天，当肌管刚开始出现纤颤时，球状和不对称形式的AChE均已存在。在第5天至第8天之间，4S（G1）、10S（G4）和16S（A12）形式急剧增加，并且出现了相当数量的12.5S（A8）AChE。当在第4天添加河豚毒素以防止纤颤时，A12和A8形式的增加被阻止，G4和G1形式的增加也受到显著损害。在第8天，发生纤颤的肌管中A12 AChE的含量比未发生纤颤的肌管多19倍，G1和G4酶的含量则多4倍以上。河豚毒素的作用是可逆的。当在第7天去除河豚毒素时，纤颤迅速恢复，球状和不对称形式也得以恢复。对发生纤颤和未发生纤颤的肌管进行光学显微镜检查表明，河豚毒素不影响肌管的总体形态发育。用O-乙基-S2-二异丙基甲基硫代磷酸酯滴定AChE活性位点表明，与纤颤相关的AChE活性增加是由于存在的AChE分子数量增加，而不是由于单个AChE分子周转乙酰胆碱的速率增加。为了评估发生纤颤和未发生纤颤的肌管中AChE的代谢情况，我们在用对氧磷使其失活后对该酶进行了检测。对氧磷很容易穿透细胞，并使AChE活性位点中的丝氨酸二乙基磷酸化，从而使其失活。二乙基磷酸化的酶是稳定的，但可以用吡啶-2-醛肟甲基碘（2-PAM）迅速且定量地重新激活。在用对氧磷使AChE失活后，我们同时评估了合成（通过追踪新合成的活性AChE）和周转（通过追踪2-PAM可重新激活的AChE）。我们的结果表明，发生纤颤的肌管中球状和不对称形式的AChE合成速度均比未发生纤颤的肌管快。
