Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling

Ahmad Safa, R. L. Felsted

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-β-aminoethylvindesine ([3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identity 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 specifity 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 (K(a)(vinblastine)/K(a)(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 μg 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 α- or β-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.

Original languageEnglish (US)
Pages (from-to)1261-1267
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number3
StatePublished - 1987
Externally publishedYes

Fingerprint

Vinca Alkaloids
Labeling
Brain
Vinblastine
Peptides
Tubulin
4-Aminobenzoic Acid
Daunorubicin
Colchicine
Ultraviolet Rays
Bioactivity
Immunoprecipitation
Ultraviolet radiation
Detergents
N-(4-azidobenzoyl)-N'-beta-aminoethylvindesine
Monoclonal Antibodies
Association reactions
Pharmacology
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specific Vinca alkaloid-binding polypeptides identified in calf brain by photoaffinity labeling. / Safa, Ahmad; Felsted, R. L.

In: Journal of Biological Chemistry, Vol. 262, No. 3, 1987, p. 1261-1267.

Research output: Contribution to journalArticle

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abstract = "A radioactive, photoactive Vinca alkaloid, N-(p-azido-[3,5-3H]-benzoyl)-N'-β-aminoethylvindesine ([3H]NABV) with pharmacological and biological activities similar to vinblastine was synthesized and used to identity 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 specifity 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 (K(a)(vinblastine)/K(a)(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 μg 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 α- or β-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.",
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