Spies-Karotkin G, Constantinides S M
Mol Cell Biochem. 1978 Nov 16;21(3):153-60. doi: 10.1007/BF00240134.
Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2M, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50 degrees or to 20 mM ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.
将部分纯化的比目鱼肌肉（美洲拟庸鲽）甘油醛-3-磷酸脱氢酶固定在溴化氰活化的琼脂糖上。研究了固定化制剂的催化特性，以确定固定化是否会改变天然全酶的动力学性质。结果表明，固定化甘油醛-3-磷酸脱氢酶的pH活性曲线与天然酶的没有差异。NAD和甘油醛-3-磷酸的米氏常数（Km）有所改变。对该酶在有和没有NAD存在的情况下对各种失活处理的稳定性进行了表征，并与天然酶的稳定性进行了比较。当将两种形式的酶与浓度大于2M的尿素一起孵育时，失活非常迅速地发生。在0.1%胰蛋白酶中孵育60分钟，固定化甘油醛-3-磷酸脱氢酶的活性降低了45%，天然可溶性酶的活性降低了70%。当暴露于50摄氏度的温度或20 mM ATP时，固定化酶也比天然可溶性酶表现出更高的稳定性。在所有情况下，NAD要么大大降低失活速率，要么完全保护酶不被失活。
