猪肾“轻型”GGT的提纯及动力学研究

Study on purification and kinetic characteristics of the “light” form of GGT from the pig kidney

为了制备ＧＧＴ测定的标准品，研究了猪肾“轻型”γ－谷氨酰移换酶（ＧＧＴ）的提取方法及动力学特性。这种“轻型”酶的比活性达３２０ｋＵ／ｇ蛋白，几乎不含其它杂酶类，每１００ＵＧＧＴ中仅含０．２６Ｕ的乳酸脱氢酶。聚丙烯酰胺梯度凝胶电泳显示一条ＧＧＴ活性区带。其催化特性和人血清ＧＧＴ非常相似，最适ｐＨ为７．８，对供体底物Ｌ－γ－谷氨酰－３－羧基－４－硝基苯胺的米氏常数（ＫＤｍ）为０．９６ｍｍｏｌ／Ｌ，对受体底物甘氨酰甘氨酸的米氏常数（ＫＡｍ）为１２．７ｍｍｏｌ／Ｌ。

The “light”form of pig kidney GGT with specific activity of 320 kU/g of protein was purified.It was almost free of contaminating enzymes.The final product,after chromatography on hydroxyapatite column,contained only 0.26 U of lactate dehydrogenase per 100 U of GGT.Gradient gel electrophoresis showed only one band of GGT activity.The “light” form of GGT was found to be very similar to the human serum enzyme in kinetic characteristics.The value of optimum pH was 7 8.The apparent Michaelis constants for donor substrate L r glutamyl 3 carboxyl 4 nitroanilide(K D m)and aceptor substrate glycylglycine(K A m) were of 0 96 and 12 7 mmol/L,respectively.