Interactions of dynein arms with B subfibers of tetrahymena cilia : Quantitation of the effects of magnesium and adenosine triphosphate

Abstract

Tetrahymena 30S dynein was extracted with 0.5 M KCI and tested for retention of several functional properties associated with its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein-tubulin interaction that we presume are directly related to the dynein forcegenerating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2− cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP-insensitive dynein binding results from dynein interactions with non-B-tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10-6 -10-3 M) has no effect on the Mg2+ -induced binding of dynein or its release by MgATP2−, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10p, M vanadate, dynein is maximally dissociated by MgATP2− concentrations ≥1 µM with half-maximal release at 0.2 /AM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme-product complex that has a low affinity for tubulin. © 1980, Rockefeller University Press., All rights reserved.