Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine

Journal Article · Tue Jan 13 00:00:00 EST 1987 · Biochemistry; (United States)

OSTI ID:6528379

Safa, A R; Hamel, E; Felsted, R L

A photoactive, radioactive analogue of vinblastine, N-(p-azido(3,5-/sup 3/H)benzoyl)-N'-(beta-amino-ethyl)vindesine (( /sup 3/H)NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and (/sup 3/H)NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of (/sup 3/H)NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of (/sup 3/H)NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.

• Research Organization: National Cancer Institute, Bethesda, MD
• OSTI ID: 6528379
• Journal Information: Biochemistry; (United States), Vol. 1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BRAIN
BIOCHEMISTRY
VINBLASTINE
BIOCHEMICAL REACTION KINETICS
TISSUE DISTRIBUTION
AZIDES
CATTLE
COLCHICINE
PHOTOCHEMISTRY
TRITIUM COMPOUNDS
ALKALOIDS
ANIMALS
ANTIMITOTIC DRUGS
ANTIPYRETICS
AROMATICS
AZAARENES
AZOLES
BODY
CENTRAL NERVOUS SYSTEM
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHEMISTRY
DISTRIBUTION
DOMESTIC ANIMALS
DRUGS
HETEROCYCLIC COMPOUNDS
INDOLES
KINETICS
LABELLED COMPOUNDS
MAMMALS
NERVOUS SYSTEM
NITROGEN COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PYRROLES
REACTION KINETICS
RUMINANTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques