[51] Activation of dynamin GTPase activity by phosphoinositides and SH3 domain-containing proteins

This chapter describes procedures to isolate milligram quantities of dynamin-1 from bovine brain and to assay dynamin GTPase activity. The most commonly used methods to analyze GTP hydrolysis utilize radioactive nucleotide, labeled either in the α- or γ-phosphate. Production of guanosine 5'-diphosphate (GDP) can be monitored using α-labeled nucleotide and separation of GDP from GTP by thin-layer chromatography (TLC) after the reaction. Phosphate release is conveniently assayed using nucleotide labeled in the 3' position and, following the reaction, separating radioactive Pi from GTP and GDP. One of the following methods usually carries out separation: adsorption of GTP and GDP to charcoal, which is then pelleted by brief centrifugation, leaving Pi in the supernatant; or extraction of Pi into an organic phase. Although radioactive assays are more sensitive, colorimetric assays can also be used to measure the amount of released phosphate.