Rapid purification of native dynamin I and colorimetric GTPase assay.
Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recombinant SH3 domains. The method greatly enriches for dynamin I at high purity and allows for large-scale biochemical and functional studies. The second method is a nonradioactive, high-throughput colorimetric GTPase assay for dynamin activity. The approach is based on terminating incubations with EDTA and the use of malachite green for high-sensitivity detection of inorganic phosphate release. The two methods will facilitate high-throughput screens for potential dynamin inhibitors or activators.
Quan
Annie
A
Cell Signalling Unit, Children's Medical Research Institute, Sydney, Australia.
Robinson
Phillip J
PJ
D000818:Animals; D001923:Brain Chemistry; D003124:Colorimetry; D034284:Dynamin I / Q000032:analysis / Q000302:isolation & purification*; D020558:GTP Phosphohydrolases / Q000032:analysis*; D005982:Glutathione Transferase / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012756:Sheep; D018909:src Homology Domains
D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C075637:amphiphysin; D005982:Glutathione Transferase; D034284:Dynamin I; D020558:GTP Phosphohydrolases