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Journal of Cell Science, Vol 87, Issue 1 71-84, Copyright © 1987 by Company of Biologists
JOURNAL ARTICLES |
KA Suprenant and JC Marsh
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
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.
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