Rotundo R L, Fambrough D M
Cell. 1980 Nov;22(2 Pt 2):583-94. doi: 10.1016/0092-8674(80)90368-2.
We have found that approximately one third of the total cell-associated acetylcholinesterase (AChE) is located on the plasma membrane of cultured chick embryo muscle, the remaining two thirds being found within the cells. This cell surface AChE appears to be an integral membrane protein. The surface enzyme is synthesized by the muscle cells in culture and is transported over a 2-3 hr period to the plasma membrane, where it accumulates at the rate of 2-3% of total surface AChE per hour. Once on the plasma membrane the AChE molecules are degraded by a process that exhibits first-order decay kinetics with a half-life of about 50 hr. Under the same experimental conditions, the acetylcholine receptor, a well described muscle cell integral membrane protein, has a half-life of approximately 19 hr. These studies provide the first direct evidence that the numbers of different muscle plasma membrane glycoprotein molecules are determined not only by differential rates of biosynthesis but also by differential rates of degradation. The intracellular AChE constitutes a rapidly turning-over pool of molecules. The rate of synthesis of AChE in culture is approximately 20% of the total cell-associated enzyme per hour, most of which is destined for secretion into the medium. Only a small portion of the newly synthesized AChE is retained on the plasma membrane. The time from synthesis to release of the enzyme is 2-3 hr. Using 3H-DFP to label the newly synthesized AChE, we can also show a quantitative transfer of AChE molecules from the intracellular to the extracellular compartments without any detectable residence time on the plasma membrane. By studying the synthesis transport and externalization of AChE we have defined the intracellular transport pathway and metabolic requirements for secretion in cultured muscle cells. These studies form the basis for a comparison of the metabolism of membrane-bound and secreted glycoproteins from this cell type.
我们发现，与细胞相关的乙酰胆碱酯酶（AChE）总量中约三分之一位于培养的鸡胚肌肉的质膜上，其余三分之二则存在于细胞内。这种细胞表面的AChE似乎是一种整合膜蛋白。表面酶由培养中的肌肉细胞合成，并在2 - 3小时内转运至质膜，以每小时占总表面AChE 2 - 3%的速率积累。一旦到达质膜，AChE分子就会通过一个呈现一级衰变动力学且半衰期约为50小时的过程被降解。在相同的实验条件下，乙酰胆碱受体是一种已被充分描述的肌肉细胞整合膜蛋白，其半衰期约为19小时。这些研究提供了首个直接证据，表明不同肌肉质膜糖蛋白分子的数量不仅由生物合成的差异速率决定，还由降解的差异速率决定。细胞内的AChE构成了一个快速周转的分子池。培养中AChE的合成速率约为每小时与细胞相关的总酶量的20%，其中大部分注定会分泌到培养基中。新合成的AChE只有一小部分保留在质膜上。从酶合成到释放的时间为2 - 3小时。使用³H - DFP标记新合成的AChE，我们还可以显示AChE分子从细胞内到细胞外区室的定量转移，且在质膜上没有任何可检测到的停留时间。通过研究AChE的合成、转运和胞外分泌，我们确定了培养的肌肉细胞中细胞内转运途径和分泌的代谢需求。这些研究为比较这种细胞类型的膜结合糖蛋白和分泌糖蛋白的代谢奠定了基础。
