龙虾肌甘油醛-3-磷酸脱氢酶及其衍生物与NAD~+的结合

平衡柱层析法测得每分子龙虾肌羧甲基化甘油醛-3磷酸脱氢酶能结合3.9分子NAD~+,而每分子光照酶则只能结合2分子NAD~+。 由蛋白荧光淬灭法得到,在25℃、pH7.0的磷酸盐缓冲液中,全酶、羧甲基酶及光照酶与NAD~+结合时均呈负协同性。

The Binding of NAD~+ to Lobster Muscle Glyceraldehyde-3 Phosphate Dehydrogenase and its Derivatives.

Measurments of the quenching of the intrinsic protein fluorescence of the lobster muscle glyceraldehyde-3-phosphate dehydrogenase during titration with NAD+ of the native, carboxymethylated and the irradiated enzyme carrying the fluorop hore showed that all 3 forms of the enzyme binding NAD+ with negative cooperativity. Gel permeation studies gave the results that the carboxymethylated enzyme binds 4 molecules of NAD+ per tetrameric enzyme whereas the irradiated enzyme binds only 2. The latter is in accord with the results shown in the previous paper that the irradiated lobster enzyme already has 2 molecules of a fluorescent NAD derivative in the NAD+ binding sites of 2 of the subunits. During the entire course of the preparation of the lobster enzyme the pH has been kept below 7, hence the negative cooperativity of the binding of NAD+ to the native as well as to the carboxymethylated enzymes cannot be attributed to slight damage of the enzyme molecule in alightly alkaline medium as has been suggested for the rabbit muscl enzyme.