Properties of Tubulin in Unfertilized Sea Urchin Eggs. Quantitation and Characterization by the Colchicine-Binding Reaction

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-bind...

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Bibliographic Details
Published in:The Journal of cell biology 1976-06, Vol.69 (3), p.599-607
Main Authors: Pfeffer, Thomas A., Asnes, Clara F., Wilson, Leslie
Format: Article
Language:English
Subjects:
Animals
Binding, Competitive
Biochemistry
Cells
Chicks
Colchicine - analogs & derivatives
Colchicine - metabolism
Crystals
Eggs
Embryos
Female
Glycoproteins - metabolism
Kinetics
Microtubules
Mitotic spindle apparatus
Ova
Ovum - metabolism
Podophyllotoxin - pharmacology
Sea Urchins
Structure-Activity Relationship
Sulfates
Temperature
Tubulin - metabolism
Vinblastine - pharmacology
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Summary:The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8°C and 37°C, and the thermodynamic parameters of the reaction were calculated. Δ H0=6.6 kcal/mol, Δ S0=46.5 eu, and, at 13°C, Δ G = -6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37°C. Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.69.3.599