CHOLINE ACETYLTRANSFERASE

本项目的总体目标是阐明其作用机制。 胆碱乙酰转移酶的体外活性，并确定在多大程度上 这些研究与酶在体内的行为有关。改进 亲和层析法纯化人胎盘组织中的酶 该酶的层析和物理特性包括 将继续研究亚基结构的确定。此外，动力学 盐类作用机理及对酶活性影响的研究 将通过测量平衡和溶剂的同位素交换来进行 氚的同位素效应。该酶的体外性质将是 与原位酶的性质相比，希望能更紧密地 接近活体条件。这些后一项研究将涉及使用 通透性突触体作为一种载体将酶保留在 与体内发现的大分子环境相似。这个系统将会 使pH、离子强度和底物浓度变化，而 维持突触体内的酶。 预计体外和原位研究相结合概述了 将提供一个更好的理解动力学行为 胆碱乙酰转移酶，从而使我们更好地理解 乙酰胆碱的体内合成机制。

The overall objective of this project is elucidate the mechanism of action of the enzyme choline acetyltransferase in vitro, and to determine to what extent these studies relate to the behavior of the enzyme, in vivo. Improved purification of the enzyme from human placenta by the use of affinity chromatography and physical characterization of the enzyme including determination of subunit structure will be pursued. In addition, kinetic studies on the mechanism of action and the effect of salts on enzyme activity will be conducted by measuring isotope exchange at equilibrium and solvent deuterium isotope effects. The in vitro properties of the enzyme will be compared to the properties of the enzyme in situ in hopes of more closely approximating in vivo conditions. These latter studies will involve the use of permeabilized synaptosomes as a vehicle for retaining the enzyme within a macromolecular environment approximating that found in vivo. This system will enable pH, ionic strength, and substrate concentration to be varied, while maintaining the enzyme within the synaptosome. It is anticipated that the combination of in vitro and in situ studies outlined in this proposal will provide a better understanding of the kinetic behavior of choline acetyltransferase, and hence lead to a better understanding of the mechanisms of acetylcholine synthesis in vivo.