Chapter 71 Vanadate-Mediated Photolysis of Dynein Heavy Chains

Abstract

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