龙虾肌羧甲基甘油醛-3-磷酸脱氢酶NAD~+荧光衍生物的生成与性质

龙虾肌甘油醛-3-磷酸脱氢酶与兔肌酶一样,用碘代乙酸修饰后,在NAD~+存在下经紫外光照射也能形成荧光衍生物。 pH对荧光衍生物的生成和稳定性有很大影响,同时,异类离子的不同影响也很明显。 定磷分析法测定荧光衍生物上的NAD~+含量,同位素示踪法观察衍生物生成过程的脱羧,都证明此光化学反应为半位反应。

Formation and Properties of a Fluorescent NAD~+ Derivative from Carboxymethylated Lobster Glyceraldehyde-3-phosphate Dehydrogenase.

As in the case previously reported with the rabbit muscle enzyme, the carb oxymethylated lobster muscle glyceraldehyde-3- phosphate dehydrogenase also forms a similar fluorescent NAD derivative when irradiated with ultraviolet light in the presence of NAD+. The effects of pH on the formation, fluorescent intensity and stability of this fluorescent NAD derivative have been studied. In citr ate-phosphate-borate buffer, the formation, fluorescent intensity and stability of this fluorescent NAD derivative have pH optima at 7.0, 5.5 and 6.2 respectively whereas in phosphate alone, pH has very little effect on its formation, fluorescent intensity and stability within the range of pH 5-7. It appears that in aci dir solutions citrate inhibits the formation of this fluorescent NAD derivative, quenches its fluorescence emission and decreases its stability. The stoichiometry for the formation of the fluorescent derivative and the subsequent decarboxylation have been studied by phosphate analysis as well as by following the decarboxylation with the 14C-l-iodoacetate carboxymethylated enzyme. Like the rabbit muscle enzyme, both reactions are "half-of-the-sites" reactions. Unlike the rabbit muscle enzyme crystallized at pH 8.3 during the entire course of preparation of the lobster muscle enzyme, the pH has been kept below 7. This excludes the possibility that the stoichiometry for the formation of this fluorescent NAD derivative is an artifact arising from minor injuries to the enzyme molecule during crystallization at an alkaline pH.