糖基化3－磷酸甘油醛脱氢酶的分离纯化及糖基代位点的探讨

应用糖基化蛋白亲和层析技术对兔肌及人红细胞的３－磷酸甘油醛脱氢酶的分离分析表明，兔肌非糖基化ＧＡＰＤＨ的比活为１８０—２００单位，而糖基化ｇＧＡＰＤＨ的为４０—５０单位，并占该酶蛋白总量的４０％。人类红细胞糖基化ｇＧＡＰＤＨ的活力占其总活力的５５％左右。以上结果表明：哺乳动物体内存在糖基化３－磷酸甘油醛脱氢酶。由于（１）糖基化明显影响ＧＡＰＤＨ的活力；（２）糖基化酶活性部位的巯基（Ｃｙｓ－１４９）空间位置发生了改变；（３）糖基化影响活性部位的空间构象及（４）ＯＰＴ对糖基化及非糖基化酶的修饰无论在动力学上还是在ＫＩ淬灭时都有明显差异，因此，糖基化的位点可能与赖氨酸残基有关，并且接近或位于酶的活性部位。

ISOLATION OF GLYCATED AND NON-GLYCATED D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND DETECTION OF GLYCATION

Isolation of the glycated and non-glycated D-glyceraldehyde-3-phosphate dehydrogenase from rabbit musde and human erythrocytes by affinity chromatoglaphy showed that the specific activity of the non-glycated GAPDH from rabbit muscle was 180-200 units,and that of the glycated GAPDH was 40-50 units. The glycated GAPDH was about 40% of the total GAPDH of rabbit muscle. The activity of the glycated GAPDH of human erythrocyted was 55% of the total activity in vitro. It was suggestal that the glycated GAPDH exists in mammals. The glycated sites may be near the active site of the enzyme and correlate with Lys residue, hecause (1) glycation in vitro reduces the activity markedly, (2) the conformation of active site is changed, (3) glycation leads to a change in the spatial position of the active thiols and (4) properties between the derivatives of the glycated and non-glycated GAPDH-OPT are differnet in both modification and quenching of the fluorescence by KI.