The Active GTP- and Ground GDP-Liganded States of Tubulin Are Distinguished by the Binding of Chiral Isomers of Ethyl 5-Amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl Carbamate

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• 作者：Pascale Barbier ; Vincent Peyrot ; Daniel Leynadier and ; and José ; Manuel Andreu
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：January 13, 1998
• 年：1998
• 卷：37
• 期：2
• 页码：758 - 768
• 全文大小：204K
• 年卷期：v.37,no.2(January 13, 1998)
• ISSN：1520-4995

文摘

NSC 613862 (S)-(-) and NSC 613863(R)-(+) are the two chiral isomers ofethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate. Both compounds bind to tubulin ina region that overlaps the colchicine site. They induce formationof abnormal polymers from purifiedGTP-Mg-tubulin, the active assembly form of tubulin, in glycerol-freebuffer with magnesium [De Ines,C., Leynadier, D., Barasoain, I., Peyrot, V., Garcia, P., Briand, C.,Rener, G. A., and Temple, C., Jr.(1994) Cancer Res. 54, 75-84]. In this study, weobserved that the S-isomer can promotepolymerizationof GDP-tubulin, the inactive assembly-incompetent form of tubulin, intononmicrotubular structures at acritical protein concentration of 1 mg/mL (12 mM MgCl₂).Neither the R-isomer nor colchicine havethis ability. By electron microscopy, these tubulin polymersshowed the same poorly defined filamentousstructure when GDP-tubulin or GTP-Mg-tubulin were used. By HPLCmeasurements, we demonstratedthat a dissociated GTP hydrolysis and exchange of nucleotide occurredduring the isomer-induced abnormalassembly. Both isomers inhibited the Mg²⁺-inducedtubulin self-association leading to 42 S double ringformation from GTP-Mg-tubulin or GDP-tubulin. Measurement of theirbinding under nonassociationconditions revealed a 3-fold decrease in the apparent equilibriumbinding constant of the R-isomer toGDP-tubulin relative to GTP-Mg-tubulin. For theS-isomer, the decrease in the binding constantwasless pronounced. Binding data, analyzed in terms of a system oflinked conformational and associationequilibria, provide evidence that the active ("straight") ratherthan the inactive ("curved") conformationof tubulin differentially recognizes these ligands. Whereasbinding of colchicine to tubulin is well-knownto induce GTP hydrolysis, this is the first case in which theinteraction of a ligand with the colchicine siteis shown to be sensitive to the presence of GDP or GTP at the distantnucleotide binding site.