We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 microM, and a Bmax of 3.4 x 10(-7)M ml-1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine. Microtubule assembly studies and SDS-polyacrylamide gel electrophoresis revealed that at 20-60 microM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (A350) studies further demonstrated that 20-60 microM-estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.
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