尿液中α-淀粉酶的提纯及性质研究

Study on purification and properties of α amylase from human urine

为了制备淀粉酶测定的参考品，从正常人尿液中提取了α－淀粉酶（Ａｍｙ）并对其特性进行了研究。该提制品淀粉酶的比活性达８４．３ｋＵ／ｇ蛋白，几乎不含其它杂酶（测不到ＡＬＴ、ＡＳＴ、ＬＤ、ＣＫ、ＧＧＴ、ＣｈＥ、ＡＬＰ活性浓度）。ＳＤＳ－聚丙烯酰胺凝胶电泳显示一条Ａｍｙ区带。其催化特性和人血清Ａｍｙ非常相似，用亚乙基封闭的Ｇ７－ｐＮＰ（ＥＰＳ）作底物，偶联多功能α－葡萄糖苷酶测定其米氏常数为０．１８９ｍｍｏｌ／Ｌ。利用琼脂糖凝胶电泳，碘－淀粉显色，该提取物至少被分为Ｓ２、Ｐ２两条活性区带。

For the purpose of preparing the reference material of α Amylase,the purification and properties of Amy in human urine were studied.The purified Amy has a specific activity of 84 3 kU/g of protein.It was almost free of contaminating enzymes.After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate,only one band of Amy could be seen.The final purified Amy was found to be very similar to the human serum Amy in catalytic properties.The apparent Michaelis constant of the purified Amy was 0 189mmol/L with ethylidence blocked G 7 pNP(EPS)as substrate and with multifunctional α glucosidase as auxiliary enzyme.Agarose gel electrophoresis was performed and isoenzymes were developed with iodine starch method.At least two distinct enzymatic bands,S 2 and P 2 were found.This research is intended to be a fundament to prepare a reference material for the measurement of α amylase.