Dependency of microtubule-associated proteins (MAPs) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules.
Microtubule-associated proteins (MAPs) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6 M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution. The addition of estramustine phosphate to microtubules reconstituted of MAPs prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4 degrees C was dependent on intact bindings between the tubulin and MAPs.
Fridén
B
B
Department of Zoophysiology, University of Göteborg, Sweden.
Wallin
M
M
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004961:Estramustine / Q000378:metabolism / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D017239:Paclitaxel; D014404:Tubulin / Q000378:metabolism*
D000470:Alkaloids; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D004961:Estramustine; D017239:Paclitaxel