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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Köpke, A. K. Dautzenberg, F. M. Rühmann, A. Spiess, J. |
| Description | Author Affiliation: Rühmann A ( Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Göttingen, Germany.); |
| Abstract | A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors. |
| ISSN | 00278424 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 20 |
| Volume Number | 93 |
| Language | English |
| Publisher | National Academy of Sciences |
| Publisher Date | 1996-11-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Affinity Labels Chemical Synthesis Azirines Chemistry Corticotropin-Releasing Hormone Receptors, Corticotropin-Releasing Hormone Metabolism Animals Binding, Competitive Cell Membrane Cyclic AMP Glycosylation Photochemistry Tumor Cells, Cultured Multidisciplinary |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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