Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules.
Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002417:Cattle; D004398:Dyneins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology*
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins