Hsu L L, Chao L P
J Neurosci Res. 1982;7(2):155-62. doi: 10.1002/jnr.490070207.
In order to further characterize the reaction mechanism of brain ChAc in its purified form, we have investigated the reverse reaction of ChAc in terms of pH optimum, salt effects, and substrate kinetics using a radiochemical assay. We directly measured the reaction product acetylcoenzyme A which was separated from the substrate ACh by a cation exchange column. Dowex 50W-X8 (Na+ form). The reverse reaction of ChAc was linear with incubation time up to 40 minutes, and with enzyme protein concentration up to 5 micrograms. It had a pH optimum at 7.0. At 0.22 M the monovalent chloride and bromide salts activated the reverse ChAc activity by 23-47% but the fluoride and iodide salts inhibited the reverse enzyme activity by 10-30%. Kinetic studies in the absence of salt showed that KACh was 0.62 +/- 0.06 mM, KCoA . SH was 12.68 +/- 1.21 microM, and Vmax was 11.6 +/- 1.0 nmol AcCoA/mg protein/min. These data are in disagreement with the values reported on partially purified ChAc from bovine brain by Glover and Potter [1971] and Hersh [1980]. This indicates that further investigations are necessary to clarify or resolve these differences.
为了进一步阐明纯化形式的脑乙酰胆碱酯酶（ChAc）的反应机制，我们使用放射化学分析法，从最适pH、盐效应和底物动力学方面研究了ChAc的逆反应。我们直接测量了反应产物乙酰辅酶A，它通过阳离子交换柱（Dowex 50W-X8，钠型）与底物乙酰胆碱（ACh）分离。ChAc的逆反应在长达40分钟的孵育时间内以及高达5微克的酶蛋白浓度下呈线性。其最适pH为7.0。在0.22 M时，一价氯化物和溴化物盐使ChAc的逆反应活性提高了23% - 47%，但氟化物和碘化物盐使逆酶活性降低了10% - 30%。在无盐条件下的动力学研究表明，乙酰胆碱的米氏常数（KACh）为0.62±0.06 mM，辅酶A - 巯基（KCoA.SH）为12.68±1.21 microM，最大反应速度（Vmax）为11.6±1.0 nmol乙酰辅酶A/毫克蛋白/分钟。这些数据与Glover和Potter [1971]以及Hersh [1980]报道的从牛脑部分纯化的ChAc的值不一致。这表明需要进一步研究以澄清或解决这些差异。
