Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling.
A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-beta-aminoethylvindesine [( 3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identify specific Vinca alkaloid macromolecular interactions in calf brain homogenate by photoaffinity labeling. The most prominent photolabeled species were 54.3- and 21.5-kDa polypeptides. The Vinca alkaloid-binding specificity of these polypeptides was confirmed by competitive blocking of specific photolabeling by vinblastine but not by colchicine or daunorubicin. The 54.3- and 21.5-kDa polypeptides exhibited specific half-maximum saturable photolabeling at 2.1 and 0.95 X 10(-7) M [3H]NABV, respectively. Relative vinblastine and NABV association constants (Ka vinblastine/Ka NABV) for the 54.3- and 21.5-kDa polypeptides were estimated to be 0.86 and 1.4, respectively. The 54.3-kDa component was found in both high speed (100,000 X g; 1 h) pellet and supernatant fractions, whereas the 21.5-kDa component was located primarily in the high speed pellet. Photolabeling of both components was maximal after 12-min UV light exposure, linear up to 120 micrograms of homogenate protein and only slightly affected by the nitrene scavenger p-aminobenzoic acid. The 54.3-kDa polypeptides of [3H]NABV-photolabeled calf brain high speed supernatant and detergent-solubilized high speed pellet fractions were identified as tubulin subunits by immunoprecipitation with monoclonal antibodies to alpha- or beta-tubulin subunits. Although the identity and function of the 21.5-kDa polypeptide is not known, this polypeptide may have a role in membrane-related effects of the Vinca alkaloids. These results demonstrate that [3H]NABV is an attractive tool for identifying and characterizing specific high affinity vinblastine cellular polypeptide acceptors which may initiate or mediate known and unknown mechanisms of Vinca alkaloid action.
D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D003078:Colchicine / Q000494:pharmacology; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D010777:Photochemistry; D014404:Tubulin / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology; D014748:Vinca Alkaloids / Q000378:metabolism*; D014751:Vindesine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D014404:Tubulin; D014748:Vinca Alkaloids; C048694:N-(4-azidobenzoyl)-N'-beta-aminoethylvindesine; D014747:Vinblastine; D014751:Vindesine; D003078:Colchicine; D010129:4-Aminobenzoic Acid