Activation of the dynein adenosinetriphosphatase by cross-linking to microtubules.
The microtubule-dynein complex consisting of 22S dynein from Tetrahymena cilia and MAP-free microtubules was subjected to treatment with various concentrations of 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide (EDC), a zero-length cross-linker, at 28 degrees C for 1 h. Following cross-linking of the microtubule-dynein complex, nearly all of the ATPase activity cosedimented with the microtubules in the presence of ATP. Electron microscopic observation by negative staining revealed that, following treatment with 1 mM EDC, the complex did not dissociate in the presence of ATP, although the dynein decoration pattern was disordered. The complex treated with 3 mM EDC exhibited normal microtubule-dynein patterns even after the addition of ATP. The ATPase activity of the microtubule-dynein complex was enhanced about 30-fold by the treatment with 1-3 mM EDC. These results indicate that the ATPase activation was caused by the close proximity of the dynein ATPase sites to the microtubules and provide further support for the functional interaction of all three dynein heads with the microtubule. The maximal specific activity was 12 mumol min-1 (mg of dynein)-1, corresponding to a turnover rate of 150 s-1, which may be the rate-limiting step at infinite microtubule concentration and may represent the maximum rate of force production in the axoneme.
Shimizu
T
T
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Marchese-Ragona
S P
SP
Johnson
K A
KA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002923:Cilia / Q000201:enzymology; D003432:Cross-Linking Reagents; D004398:Dyneins / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D013768:Tetrahymena / Q000201:enzymology*; D014404:Tubulin / Q000378:metabolism*
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D014404:Tubulin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins; D005022:Ethyldimethylaminopropyl Carbodiimide