Two distinct isoforms of sea urchin egg dynein.
Extracts 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 Chemistry 263:6759-6771, 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-2 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.
Grissom
P M
PM
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347.
Porter
M E
ME
McIntosh
J R
JR
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D004398:Dyneins / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D043583:Nucleoside-Triphosphatase; D009865:Oocytes / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011755:Pyrophosphatases; D012617:Sea Urchins / Q000201:enzymology*
D007527:Isoenzymes; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; C019950:CTPase; D011755:Pyrophosphatases; D043583:Nucleoside-Triphosphatase; D004398:Dyneins