| Autor: |
McCullough C; Department of Chemistry, Marquette University, Milwaukee, WI, USA., Pullela PK, Im SC, Waskell L, Sem D |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2013; Vol. 987, pp. 51-9. |
| DOI: |
10.1007/978-1-62703-321-3_4 |
| Abstrakt: |
The cytochromes P450 (CYPs) play a central role in a variety of important biological oxidations, such as steroid synthesis and the metabolism of xenobiotic compounds, including most drugs. Because CYPs are frequently assayed as drug targets or as anti-targets, tools that provide confirmation of active-site binding and information on binding orientation would be of great utility. Of greatest value are assays that are reasonably high throughput. Other heme proteins, too-such as the nitric oxide synthases (NOSs), with their importance in signaling, regulation of blood pressure, and involvement in the immune response-often display critical roles in the complex functions of many higher organisms, and also require improved assay methods. To this end, we have developed an analog of cyanide, with a (13)CH3-reporter group attached to make methyl isocyanide. We describe the synthesis and use of (13)C-methyl isocyanide as a probe of both bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. The (13)C-methyl isocyanide probe can be used in a relatively high-throughput 1-D experiment to identify binders, but it can also be used to detect structural changes in the active site based on chemical shift changes, and potentially nuclear Overhauser effects between probe and inhibitor. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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