| Autor: |
Allaire PD; Department of Biology, University of Utah, Salt Lake City, UT, 84112, USA., McPherson PS, Ritter B |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2015; Vol. 1298, pp. 217-31. |
| DOI: |
10.1007/978-1-4939-2569-8_19 |
| Abstrakt: |
Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that directly measures the exchange activity of DENN domains by monitoring loading of GTP onto a Rab GTPase. Rabs are first purified from bacterial or mammalian sources and are then loaded with GDP. Purified DENN domains or DENN domain-bearing proteins are added in the presence of [(35)S]GTPγS and the transfer of [(35)S]GTPγS to the Rab is measured by filtering the reaction over nitrocellulose membranes to trap the Rab and thus the associated [(35)S]GTPγS. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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