title: Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus.
abstract: Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D012617:Sea Urchins; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D014404:Tubulin