Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP
Department of Cell Biology
Adenosine Triphosphatases; Adenosine Triphosphate; Affinity Labels; Azides; Binding Sites; Binding, Competitive; Chlamydomonas; Dynein ATPase; Electrophoresis, Polyacrylamide Gel; Kinetics; Molecular Weight; Photochemistry; Ultraviolet Rays; Vanadates; Vanadium
The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, label is incorporated primarily into the alpha, beta, and Mr = 78,000 chains; a much smaller amount is incorporated into the Mr = 69,000 chain. The light chains are not labeled. The incorporation is UV-dependent, ATP-sensitive, and blocked by preincubation of the enzyme with vanadate plus low concentrations of ATP or ADP. These results suggest that the alpha heavy chain contains the site of ATP binding and hydrolysis in the alpha subunit. In the beta subunit, the beta heavy chain and one or both intermediate chains may contain ATP-binding sites.
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Citation: J Biol Chem. 1985 Oct 15;260(23):12844-50.
The Journal of biological chemistry
Pfister, K. Kevin; Haley, Boyd E.; and Witman, George B., "Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP" (1985). Cell and Developmental Biology Publications and Presentations. Paper 61.