Mechanism of assembly of sea urchin egg tubulin.
Tubulin purified from eggs of the sea urchin Strongylocentrotus purpuratus assembles efficiently in vitro to form microtubules at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. MAPs, ring oligomers, and high concentrations of nonphysiological solvents are not required for the assembly reaction. At concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C a concentration-dependent overshoot in turbidity and in light scattering at small angles was observed: turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Electron microscopic analysis demonstrated that tubulin sheets were prevalent during the initial stages of overshoot assembly, whereas complete microtubules were present at steady state. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. The overshoot cannot be explained by proteolysis or denaturation of tubulin, by depletion of GTP, by a decrease in assembled mass, or by redistribution of polymer lengths. Taken together, the results suggest that changes in the form and/or in the organization of the assembling polymer are responsible for the overshoots in turbidity and in light scattering at small angles. Our results are consistent with models of microtubule assembly that postulate nucleation by tubulin sheets and subsequent folding of the sheets to form mature microtubules.
Detrich
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Jordan
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D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D010063:Ovum / Q000378:metabolism* / Q000648:ultrastructure; D012617:Sea Urchins; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D014404:Tubulin; D006160:Guanosine Triphosphate