A microtubule-activated ATPase from sea urchin eggs, distinct from cytoplasmic dynein and kinesin.

Abstract

We report an ATPase activity, present in sea urchin egg cytosol, that is activated by microtubules. The activity sediments at 10 S in sucrose gradients and is clearly distinct from activities at 12 S and 20 S due to cytoplasmic dynein. Potent activation of the ATPase is observed when endogenous egg tubulin is induced to assemble with taxol or when exogenous taxol-stabilized pure brain tubulin microtubules or flagellar outer-doublet microtubules are added. No activation by tubulin subunits or taxol alone is detectable. In contrast to flagellar or cytoplasmic dynein, the microtubuleactivated enzyme is unaffected by vanadate or by nonionic detergents and hydrolyzes GTP in addition to ATP. In contrast to kinesin, it cosediments with microtubules in the presence or absence of ATP. The microtubule-activated enzyme may have a role in microtubule-based motility.

Characterization Of The Sea-Urchin Egg Microtubule-Activated Atpase

“…Addition of exogenous microtubules to egg cytosolic extracts led to an activation of the A T P ase activity similar to that induced by taxol (Collins & Vallee, 1986). M icrotubules prepared from calf brain tubulin and flagellar axoneme outer doublet microtubules were equally effective in activating the A TPase.…”

“…Further activation was not observed in the presence of higher taxol concentrations. In other experiments, the microtubules formed in the presence of increasing taxol concentrations were sedimented from the cytosolic extract (Collins & Vallee, 1986). All the newly induced activity was found to cosediment with the microtubules.…”

“…Effect of additional microtubules on cytosolic ATPase activity. Pure calf brain tubulin was assembled with taxol (as described by Collins & Vallee, 1986) and added at the concentrations indicated to samples of the same extracts shown in A previously treated with 10 UM-taxol to assemble endogenous tubulin. After a 5 min incubation at 37°C, the extracts were assayed for ATPase activity.…”

“…Taxol was added at the concentrations indicated to a cytosolic extract prepared from eggs of the sea-urchin Strongylocentrotus purpuratus (Collins & Vallee, 1986). After incubation at 37°C for 5 min to assemble microtubules, the treated extracts were assayed for ATPase activity (Collins & Vallee, 1986). B.…”

“…The effect of microtubules on enzymic activity was determined by addition to a final concentration of 0-25 mg ml-1 of purified calf brain tubulin that had been assembled into microtubules with taxol. Gradient fractions were assayed for ATPase activity at 30 °C in the presence (O-----O) or absence ( • -----• ) of microtubules (from Collins & Vallee, 1986). and addition of exogenous microtubules was more than twofold over the untreated control.…”