AbstractExtracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr‐3, binds to microtubules in an ATP‐sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.:Journal of Biological Chemistry263:6759–6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr‐3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60 of its MgATPase activity. Non‐ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr‐3. Sucrose gradient‐purified HMr‐3 promotes an ATP‐sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr‐3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr‐3 is related to the dynein‐like enzymes isolated from a variety of cell types and may represent the cytoplasmic dy
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