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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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Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1976
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2109704/
https://www.ncbi.nlm.nih.gov/pubmed/944700
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collection PubMed
description 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 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). 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.
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spelling pubmed-21097042008-05-01 Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction J Cell Biol Articles 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 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). 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. The Rockefeller University Press 1976-06-01 /pmc/articles/PMC2109704/ /pubmed/944700 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title_full Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title_fullStr Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title_full_unstemmed Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title_short Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction
title_sort properties of tubulin in unfertilized sea urchin eggs. quantitation and characterization by the colchicine-binding reaction
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2109704/
https://www.ncbi.nlm.nih.gov/pubmed/944700