糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

DETERMINATION OF THE SUGAR BOUND TO D-GLYCERADEHYDE-3-PHOSPHATE DEHYDROGENASE AND STUDY ON CONFORMATIONAL CHANGES OF THE ENZYME

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小，但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明，ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变，糖基化位点可能发生在酶活性部位附近。

The determination of the sugar bound to GAPDH and investigation of conformational changes of the glycatal and non-glycated GAPDH have been performed by methods of orcinol sulfuric acid, phenol sulfuric acid, CD, fluorescence and DTNB modifying.The glycatal GADPH which purified by the affinity chromatography of m-aminophenylboronic acid linked to Sepharose CL 6B should consist of 1.89 glucose.The cirvular dichroism spectra between gGAPDH and GPADH were appreciable different in the near ultraviolet region, but less in the far ultraviolet region. That changes in intrinsic fluoresence between the two.enzyme are distinct in GuHCl solutions of different concentrations were also observed. The kinetic of modification of the nonglycated enzyme was a biphasic proedure which can be divided into fast and slow phase. But that of the gGAPDH is a triphasic one which may be divided into fast, luteinte and slow phase. The fast phase of gGAPDH is faster than that of GAPDH during DTNB modification. It is suggestal that glycation of enzyme might lead to some changes in conformation of the active site and the molecule as a whole,the glycation might occur at Lys residues which may be in situ at or near the active site.