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Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2′,5′-ADP-Sepharose pull-down assay

Author:
Long, Yang, Yan, Jianghong, Luo, Suxin, Liu, Zhenguo, Xia, Yong
Source:
Analytical biochemistry 2017 v.537 pp. 8-12
ISSN:
0003-2697
Subject:
N-acetylglucosamine, animal disease models, animal tissues, antibodies, binding capacity, cultured cells, diabetes, endothelial cells, endothelial nitric oxide synthase, precipitin tests, rats, resins, vascular tissues
Abstract:
Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2′,5′-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2′,5′-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.
Agid:
5830849