Ali M, Brownstone Y S
Biochim Biophys Acta. 1976 Aug 12;445(1):74-88. doi: 10.1016/0005-2744(76)90161-3.
The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.
通过对吉田和渡边（1972年，《生物化学杂志》247卷，440 - 445页）方法的改进，已从人红细胞中分离出结晶形式的ATP - 3 - 磷酸 - D - 甘油酸 - 1 - 磷酸转移酶（EC 2.7.2.3）（磷酸甘油酸激酶）。使用载体两性电解质（pH 7 - 9）通过电聚焦进一步纯化结晶酶。磷酸甘油酸激酶的等电点估计为8.75。在pH 8.3（37℃）下，经电聚焦纯化的磷酸甘油酸激酶的比活性为每毫克蛋白质2200单位。使用荧光法测定与NAD偶联的酶，以测量极低底物浓度下的反应速率，从而在从1,3 - 二磷酸甘油酸到3 - 磷酸甘油酸的正向方向上测定酶活性。过量添加辅助指示酶以产生真正的初始速度动力学，即在添加底物（1,3 - 二磷酸甘油酸）时没有时间滞后。这在理论上使用数学模型得以确立并通过实验得到证实。当改变测定条件时，进一步证明磷酸甘油酸激酶是限速步骤。
