Steady-state kinetic study of vanadate-induced inhibition of ciliary dynein adenosinetriphosphatase activity from Tetrahymena.

Inhibitory action of vanadate (orthovanadate and metavanadate) on ciliary dynein adenosinetriphosphatase (ATPase) from Tetrahymena was investigated. The apparent concentrations of vanadate giving half-maximal inhibition of Mg-ATPase activity of various dynein fractions were as follows: the axoneme-bound form of dynein at 100 nM, solubilized crude dynein at 50 nM, 14S dynein at 5 microM, and 30S dynein at 20 nM. The Ca-ATPase of 30S dynein was more than 30-fold less sensitive than its Mg-ATPase, and still less sensitive was the Ca-ATPase of 14S dynein. The Mg-ATPase of 30S dynein was most sensitive to vanadate at neutral pH, and the addition of KCl or NaCl into the assay mixture reduced its sensitivity. Varying the assay temperature between 0 and 37 degrees C affected the sensitivity to a slight extent. Metavanadate was as much a potent inhibitor of dynein ATPase as orthovanadate, but vanadium pentoxide was less potent. When the dynein ATPase activity was reciprocally plotted against the concentration of vanadate (the Dixon plot), the inhibition was proved to be biphasic. At lower concentrations of vanadate, the inhibition was more significant. Therefore the Dixon plot had a downward bent. Reexamination of the Lineweaver-Burk plot of 30S dynein Mg-ATP showed a downward bent, which indicates that 30S dynein may have at least two Km values, ca. 1 microM and 3 microM; or otherwise, 30S dynein might possibly have a negatively cooperative nature (Hill coefficient 0.67). The vanadate-induced inhibition of 30S dynein Mg-ATPase was noncompetitive in the entire range of ATP concentration examined. Since the vanadate-induced inhibition of 30S dynein Mg-ATPase could be classified into "tight-binding inhibition", we could estimate the dissociation constant of vanadate and the molecular weight per enzymatic active site according to the kinetics of tight-binding inhibition with several assumptions. Thus, the dissociation constant was 10-15 nM, depending on the ATPase assay condition, while the molecular weight per enzymatic active site was 420 000-480 000, independent of the assay condition with the assumption that the present 30S dynein preparation is totally pure. This value would be reduced about 20% when the purity was taken into consideration.