一种用于粗细胞提取物中新霉素磷酸转移酶定性和定量测定的灵敏新方法。

A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.

作者信息

Reiss B, Sprengel R, Will H, Schaller H

出版信息

Gene. 1984 Oct;30(1-3):211-7. doi: 10.1016/0378-1119(84)90122-7.

Abstract

A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

摘要

描述了一种检测和定量粗细胞提取物中低含量新霉素磷酸转移酶的通用方法。该测定基于酶与其他干扰蛋白的电泳分离，并通过抗生素卡那霉素的原位磷酸化检测其酶活性。作为底物的卡那霉素和[γ-32P]ATP都嵌入到放置在含有分离蛋白的聚丙烯酰胺凝胶上的琼脂糖凝胶中。酶促反应后，将磷酸化的卡那霉素转移到P81磷酸纤维素离子交换纸上，并通过放射自显影观察放射性标记的卡那霉素。用这种方法可以很容易地检测到1 ng活性酶。原核和真核细胞提取物都可以进行检测，并且可以确定酶活性蛋白大小的变化。