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Anal Biochem. 2017 Nov 15;537:8-12. doi: 10.1016/j.ab.2017.08.017. Epub 2017 Aug 24.

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

Author information

1
Division of Cardiology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China.
2
Institute of Life Science, Chongqing Medical University, Chongqing, 400010, China.
3
Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, 473 West 12th Avenue, Columbus, OH, 43210, USA.
4
Division of Cardiology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China; Institute of Life Science, Chongqing Medical University, Chongqing, 400010, China; Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, 473 West 12th Avenue, Columbus, OH, 43210, USA. Electronic address: yong.xia@osumc.edu.

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.

KEYWORDS:

2′,5′-ADP-Sepharose; Endothelial nitric oxide synthase; O-linked N-acetylglucosamine; Pull-down assay

PMID:
28844813
DOI:
10.1016/j.ab.2017.08.017
[Indexed for MEDLINE]

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