Estramustine binds MAP-2 to inhibit microtubule assembly in vitro.
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.
Stearns
M E
ME
Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA 19111.
Tew
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KD
D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004961:Estramustine / Q000378:metabolism*; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000648:ultrastructure; D009588:Nitrogen Mustard Compounds / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014404:Tubulin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D009588:Nitrogen Mustard Compounds; D014404:Tubulin; D004961:Estramustine