A procedure was developed for Isolating Chlamydonzonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dyneln of this organism, containing three heavy chains (α, β, γ), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the α and β heavy chains and a 12-S particle containing the γ heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+ the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dyneln had the activity to bind to the axonemes of oda1 and Increase the reactivated motility of detergent-extracted cell models; Its addition increased the beat frequency from 28Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to Increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains Its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Biochemistry|
|State||Published - Jun 1992|