Localization of the vinblastine-binding site on beta-tubulin.
A fluorescent vinblastine derivative, vinblastine-4'-anthranilate, has been shown to inhibit polymerization of rat brain tubulin (IC50 = 4.8 microM). Binding of the drug to tubulin increases fluorescence intensity, causes a small emission blue shift, and has a quantum yield of 0.037. Fluorescence increases as a function of drug concentration, with a high affinity site and an undetermined number of lower affinity sites. Photolabeling, by exciting the fluorescent drug-tubulin complex at the absorption maximum of anthranilate, yields a covalent adduct confined to beta-tubulin. Its formation is specific in that it is blocked by maytansine or vinblastine. Tryptic hydrolysis identifies a single fluorescent beta-peptide coinciding with residues 175-213. The interactions between various ligands at this central portion of beta-tubulin are discussed.
Rai
S S
SS
Laboratory of Biochemical Pharmacology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wolff
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014357:Trypsin; D014404:Tubulin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D014747:Vinblastine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin; D062367:ortho-Aminobenzoates; C099968:vinblastine-4'-anthranilate; D014747:Vinblastine; D014357:Trypsin; D003545:Cysteine