Temperature and pH govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts.
A new method for microtubule purification from unfertilized sea-urchin eggs was developed in order to obtain large quantities of calcium- and cold-labile microtubules that contained microtubule-associated components important for mitosis. By taking into consideration the pH, ionic composition of egg cytoplasm, and the physiological temperature for growth of the Pacific coast sea-urchin Strongylocentrotus purpuratus, methods were developed for the assembly of intact microtubules directly from unfertilized egg extracts. The microtubules obtained by cycles of temperature-dependent assembly and disassembly are composed of tubulin and abundant microtubule-associated proteins. These microtubules are cold- and calcium-labile and assemble at a critical protein concentration of 0.11 mg ml-1 at 24 degrees C. The yield of microtubule protein obtained by this new method is equivalent to that obtained with taxol (6-8 mg/20 ml packed eggs). Microtubules that have been fixed and prepared for electron microscopy are decorated with large, globular projections that are attached to the microtubule by thin stalks.
Suprenant
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KA
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
Marsh
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JC
D000199:Actins / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008868:Microtubule Proteins / Q000032:analysis; D008870:Microtubules / Q000502:physiology*; D010063:Ovum / Q000032:analysis*; D012617:Sea Urchins; D013696:Temperature; D014404:Tubulin / Q000032:analysis
D000199:Actins; D008868:Microtubule Proteins; D014404:Tubulin