Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs.
Taxol was used to prepare microtubules from unfertilized eggs of sea urchins Lytechinus pictus, Strongylocentrotus droebachiensis , and Strongylocentrotus purpuratus. By electron microscopy, these microtubules possessed normal morphology and were decorated with projections. The polypeptides present were tubulin plus microtubule-associated proteins (MAPs) which included various high molecular weight polypeptides, and a Mr = 80,000 polypeptide. These MAPs were extracted from the microtubules by differential centrifugation in high ionic strength buffers, yielding a pellet of microtubules which were not decorated with projections. The Mr = 80,000 and high molecular weight MAPs were separated using Bio-Gel A-1.5 m chromatography, and shown to bind taxol-stabilized microtubules assembled from purified bovine brain tubulin. A dynein-like MgATPase activity is present in sea urchin egg extracts. 10-20% of this MgATPase co-pelleted with the taxol-assembled microtubules, under conditions where greater than 90% of the tubulin pelleted. During subsequent fractionation of the microtubules, by (i) high salt extraction followed by gel filtration or sucrose density gradient fractionation or (ii) ATP extraction, the MgATpase co-purified with high Mr MAPs. The MgATPase which remained in the microtubule-depleted egg extract was partially purified by (NH4)2SO4 fractionation, followed by Bio-Gel A-5 m and hydroxylapatite chromatography. The high Mr MAP MgATPase and the hydroxylapatite MgATPase both contained a prominent polypeptide (Mr approximately 350,000), which co-migrated on sodium dodecyl sulfate gels with the major heavy chain of dynein extracted from sperm axonemes. Our data suggest that this Mr approximately 350,000 polypeptide is cytoplasmic dynein.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase; D004398:Dyneins; D005260:Female; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000187:drug effects / Q000648:ultrastructure*; D008970:Molecular Weight; D010063:Ovum / Q000201:enzymology* / Q000648:ultrastructure; D017239:Paclitaxel; D011506:Proteins / Q000302:isolation & purification; D012617:Sea Urchins; D013045:Species Specificity
D000470:Alkaloids; D000972:Antineoplastic Agents, Phytogenic; D008869:Microtubule-Associated Proteins; D011506:Proteins; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D004398:Dyneins; D017239:Paclitaxel