CHOLINE ACETYLTRANSFERASE

本项目的总体目标是阐明 胆碱乙酰转移酶在体外，并确定在何种程度上 这些研究涉及酶在体内的行为。 改进 用亲和层析法从人胎盘中纯化酶 酶的色谱和物理表征，包括 将进行亚基结构的测定。 此外，Kinetic 盐对酶活性的影响及作用机理的研究 将通过测量平衡和溶剂下的同位素交换来进行 氘同位素效应 酶的体外性质将是 与原位酶的性质相比， 接近体内条件。 这些研究将涉及使用 透化的突触体作为将酶保留在细胞内的载体， 大分子环境近似于体内发现的环境。 该系统将 使pH、离子强度和底物浓度能够变化，同时 维持突触体内的酶。 预计体外和原位研究的结合概述了 在这个建议将提供一个更好的了解动力学行为的 胆碱乙酰转移酶，从而导致更好地了解 乙酰胆碱在体内的合成机制。

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.