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| Content Provider | PubMed Central |
|---|---|
| Author | Johnson, Gary L. Macandrew, Vincent I. Pilch, Paul F. |
| Abstract | The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach 125I-labeled glucagon covalently to a rat liver membrane protein of M r ≈ 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 M r 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 M r 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 M r 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 M r 53,000 peptide with an efficiency of 1-2% can be used to attach 125I-labeled insulin covalently to a M r 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. |
| Starting Page | 875 |
| File Format | |
| ISSN | 10916490 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 2 |
| Volume Number | 78 |
| Language | English |
| Publisher Date | 1981-02-01 |
| Access Restriction | Open |
| Subject Keyword | Research in Higher Education |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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