Direct incorporation of guanosine 5-diphosphate into microtubules without guanosine 5-triphosphate hydrolysis

Hamel, Ernest ; Batra, Janendra K. ; Lin, Chii M. (1986) Direct incorporation of guanosine 5-diphosphate into microtubules without guanosine 5-triphosphate hydrolysis Biochemistry, 25 (22). pp. 7054-7062. ISSN 0006-2960

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Official URL: http://pubs.acs.org/doi/abs/10.1021/bi00370a045?pr...

Related URL: http://dx.doi.org/10.1021/bi00370a045

Abstract

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.

Item Type:Article
Source:Copyright of this article belongs to American Chemical Society.
ID Code:13453
Deposited On:11 Nov 2010 09:14
Last Modified:17 May 2011 10:17

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