Cytoplasmic dynein-like ATPase cross-links microtubules in an ATP-sensitive manner.

We have prepared dynein-like ATPase from the eggs of the sea urchin Strongylocentrotus purpuratus using differential centrifugation and column chromatography. This ATPase preparation is inhibited by vanadate and erythro-9-(3-[2-hydroxynonyl]) adenine (EHNA) at concentrations similar to those that inhibit reactivated flagellar beating and spindle elongation in lysed cell models. Using microtubule affinity and ATP-induced release, we can purify this ATPase activity to a composition on SDS PAGE of four peptides ranging in molecular weight from 180,000-300,000. When viewed in darkfield optics, this affinity-purified ATPase caused extensive parallel bundling of microtubule-associated protein-free microtubules. These bundles were dispersed by 1 mM ATP but not by ATP gamma S or AMP-5'-adenylimidodiphosphate. The reformation of microtubule bundles after dispersal by ATP required ATP hydrolysis; bundles did not reform in the presence of 10 microM vanadate. Negative stain electron microscopy of these bundled microtubules revealed that they are arranged in parallel networks with extensive close lateral association.