Tsuchiya M, Osago H, Shimoyama M
Department of Biochemistry, Shimane Medical University, Japan.
Anal Biochem. 1995 Jan 20;224(2):486-9. doi: 10.1006/abio.1995.1076.
Using high-voltage capillary electrophoresis we detected ADP-ribosylarginine, a product of ADP-ribosylation reaction catalyzed by arginine-specific ADP-ribosyltransferase in the presence of NAD and L-arginine. The authentic ADP-ribosylarginine, detected by its ultraviolet absorbance at 254 nm, had a different retention time from NAD or nicotinamide. When the ADP-ribosylation reaction products were analyzed, the peak corresponding to ADP-ribosylarginine increased with incubation time and in an enzyme-dose-dependent manner. The lower limit of detection was 0.3 pmol, a value 100 times lower than that obtained with the reversed-phase high-performance liquid chromatography assay described previously. Using the capillary electrophoresis system, a thiol-independent ADP-ribosyltransferase activity was detected in chicken spleen cell membrane. Since the capillary electrophoresis assay for ADP-ribosylarginine is simpler, faster, and less expensive than the high-performance liquid chromatography assay, determination of arginine-specific ADP-ribosyltransferase activity in animal tissues will be facilitated.
利用高压毛细管电泳，我们检测到了ADP - 核糖基精氨酸，它是在烟酰胺腺嘌呤二核苷酸（NAD）和L - 精氨酸存在的情况下，由精氨酸特异性ADP - 核糖基转移酶催化的ADP - 核糖基化反应的产物。通过其在254 nm处的紫外吸收检测到的真实ADP - 核糖基精氨酸，其保留时间与NAD或烟酰胺不同。当分析ADP - 核糖基化反应产物时，对应于ADP - 核糖基精氨酸的峰随孵育时间增加，并呈酶剂量依赖性。检测下限为0.3 pmol，该值比先前描述的反相高效液相色谱测定法获得的值低100倍。使用毛细管电泳系统，在鸡脾细胞膜中检测到了一种不依赖硫醇的ADP - 核糖基转移酶活性。由于用于ADP - 核糖基精氨酸的毛细管电泳测定法比高效液相色谱测定法更简单、更快且成本更低，因此将有助于测定动物组织中精氨酸特异性ADP - 核糖基转移酶的活性。
