Sedimentation velocity studies of the vinca-alkaloid-induced self-association of tubulin

摘要

Abstract: Vinca alkaloids induce tubulin to self-associate into coiled spirals that further align into sheets and macrotubes. The energetics of spiral formation has been studied by sedimentation velocity in a Beckman Optima XLA analytical ultracentrifuge. The analysis process involves conversion of an absorption based sedimentation pattern into a sedimentation distribution, g(s), and determination of mechanism involving isodesmic ligand-medicated or ligand- mediated plus ligand-facilitated self-association. We have compared the vinca alkaloid-induced self-association of porcine brain tubulin in the presence of 50 $mu@M GTP or GDP. For each drug investigated the affinity is shown to be enhanced by GDP and allosterically linked to GDP/GTP, NaCl and divalent cation binding. These allosteric effectors differentially interact with one another. Thus, GDP enhances self-association over GTP by 0.90 kcal/mol, but the enhancement is reduced to 0.35 kcal/mol by increasing NaCl concentration to 150 mM. High salt stimulates spiral formation but it affects GTP-tubulin preferentially over GDP-tubulin. Divalent cations stimulate spiral formation but GDP-tubulin differentiates between Mg$+$PLU@2$/, Ca$+$PLU@2$/, and Mn$+$PLU@2$/, while GTP-tubulin does not. Divalent cations, Ca$+$PLU@2$/ and Mn$+$PLU@2$/, induce spiral condensation and macrotube formation, but this is also inhibited by high salt. The differential action of these effectors suggests an interpretation of the energetics in terms of a structural model where charge localization, binding domains and conformational interactions within and between the tubulin heterodimers are responsible for the observed allosteric effects. !9