Stabilization of Tubulin by Deuterium Oxide
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- 作者：Gopal Chakrabarti ; Shane Kim ; Mohan L. Gupta ; Jr. ; Janice S. Barton ; and Richard H. Himes
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 9, 1999
- 年：1999
- 卷：38
- 期：10
- 页码：3067 - 3072
- 全文大小：176K
- 年卷期：v.38,no.10(March 9, 1999)
- ISSN：1520-4995
文摘
Tubulin is an unstable protein when stored in solution and loses its ability to form microtubulesrapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 
C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 C, but in buffer prepared in D2O, noactivity was lost after 54 h. Tubulin was completely inactivated at 37 C in 8 h in H2O buffer, but only20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slowerrate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at bothtemperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer;however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobicsites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowedsomewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involvethe disruption of hydrophobic forces. The conformational change is followed by an aggregation processthat cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys.Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We alsoobserved that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbonstructures and incompletely folded microtubules. When these polymers were disassembled and reassembledin H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO,both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation ofribbon structures rather than microtubules.
C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 C, but in buffer prepared in D2O, noactivity was lost after 54 h. Tubulin was completely inactivated at 37 C in 8 h in H2O buffer, but only20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slowerrate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at bothtemperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer;however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobicsites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowedsomewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involvethe disruption of hydrophobic forces. The conformational change is followed by an aggregation processthat cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys.Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We alsoobserved that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbonstructures and incompletely folded microtubules. When these polymers were disassembled and reassembledin H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO,both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation ofribbon structures rather than microtubules.