期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1981
卷号：78
期号：2
页码：875-878
DOI：10.1073/pnas.78.2.875
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach 125I-labeled glucagon covalently to a rat liver membrane protein of Mr {approx} 53,000. Membranes that had been incubated with 125I-labeled glucagon were treated in the dark with hydroxysuccinimidyl-p-azidobenzoate, and a covalent complex was then formed by irradiation with ultraviolet light. Characteristics of 125I-labeled glucagon binding and covalent attachment to the Mr 53,000 peptide were consistent with this peptide being a component of the glucagon receptor involved in the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1 ]. Binding and covalent attachment of 125I-labeled glucagon to the Mr 53,000 peptide were inhibited by glucagon concentrations that were within the dose-response curve for adenylate cyclase activation, and GTP specifically decreased the photoaffinity crosslinking of 125I-labeled glucagon to the Mr 53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of 125I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached 125I-labeled glucagon to the Mr 53,000 peptide with an efficiency of 1-2% can be used to attach 125I-labeled insulin covalently to a Mr 125,000 peptide with an efficiency of approximately 10%. This peptide has been shown to be a subunit of the high-affinity insulin-binding site in rat liver membranes. The technique of photoaffinity crosslinking with agents like hydroxysuccinimidyl-p-azidobenzoate provides a rapid, simple method of covalently attaching ligands to their putative receptors. Photoaffinity crosslinking does not require chemical modification of the labeled ligand and has a less stringent requirement for specific reactive groups than the commonly used bifunctional crosslinking reagents.
关键词：hydroxysuccinimidyl- p -azidobenzoate ; covalent attachment ; hormones
