Photosensitized cleavage of dynein heavy chains. Cleavage at the V2 site by irradiation at 365 NM in the presence of oligovanadate.
Irradiation of the outer-arm dynein ATPase from sea urchin sperm flagella at 365 nm in the presence of 50-200 microM vanadate (Vi) and 1 mM manganese acetate, in the absence of ATP, cleaves the alpha and beta heavy chains at a specific site, termed the V2 site, to form discrete peptides of Mr approximately 260,000 and 170,000 from the alpha chain and of Mr approximately 255,000 and 175,000 from the beta chain, with a yield of 80%. This cleavage at the V2 site is not correlated with any direct effect on the dynein ATPase activity. In the presence of 100 microM Vi, the half-times for cleavage of the alpha and beta chains are about 12 and 50 min, respectively. The rate of heavy chain cleavage shows a sigmoidal dependence upon Vi concentration, with half-maximal rate occurring at 58 +/- 7 microM, consistent with the chromophore responsible for cleavage being tri-vanadate. Addition of 10 microM ATP or ADP, or of 100 microM CTP or UTP, to the irradiation medium inhibits cleavage at the V2 site, and results in a slow cleavage occurring at the V1 site described previously. The peptides produced by sequential cleavage at the V2 and then the V1 sites indicate that the sites are separated by about 100,000 Da along the length of each heavy chain. Photoaffinity labeling with [alpha-32P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) gives specific incorporation of 32P into both the Mr 255,000 and 175,000 peptides of the beta chain but into only the Mr 260,000 peptide of the alpha chain. These results suggest that V2 cleavage occurs on a loop of the heavy chain that forms part of the ATP-binding site, close to the locus of 8-N3ATP attachment.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D010782:Photolysis; D012617:Sea Urchins; D013997:Time Factors; D014544:Uridine Triphosphate / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology*
D000345:Affinity Labels; D001386:Azides; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins; D014544:Uridine Triphosphate