Vanadate-mediated photolysis of dynein heavy chains.

摘要: Publisher Summary Vanadate-mediated photolysis is very useful in efforts to locate specific sites within DHCs and V1 cleavage also has been used as a diagnostic tool identify presumptive cytoplasmic dyneins. Dynein heavy chains (DHCs) may be cleaved at two discrete sites, termed V2, by UV irradiation the presence of vanadate. Although both reactions involve vanadate chromophore, solution requirements for are quite different. In first, occurs ATP (or ADP) low submicromolar levels At these concentrations, remains monomeric acts phosphate analog an enzyme-ADP-vanadate complex. This reaction supported variety cations including Mg 2+ , Ca Zn but inhibited transition metals such Mn Fe Co . Interestingly, requirement nucleotide not absolute, several cases photocleavage obtained absence ATP. thought occur active site enzyme, or near region that coordinates γ nucleotide. Several lines evidence support this hypothesis. Photocleavage V2 requires higher concentrations (>∼100 μM) chapter describes basic techniques obtaining dyneins sites. These simple methods applied purified enzyme situ