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Photoaffinity derivative of colchicine: 6'-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine. Photolabeling and location of the colchicine-binding site on the alpha-subunit of tubulin

Journal Article · · J. Biol. Chem.; (United States)
OSTI ID:6182075
; ; ; ; ; ; ;
A photoaffinity analog of colchicine, 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine, was synthesized by reacting deacetylcolchicine or (TH)deacetylcochicine with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Homogeneity of the photoaffinity analog was established by thin-layer chromatography and high-pressure liquid chromatography. The structure of the photoaffinity analog was determined by H and TC NMR, infrared and ultraviolet-visible spectroscopies, and elemental analysis. Binding of 6-(4'-azido-2'-nitrophenylamino)hexanoyldeacetylcolchicine to bovine renal tubulin was measured by competition with (TH)colchicine. The value of the apparent Ki for the photoaffinity analog was 0.28 microM in the concentration range of 0.8-1.2 microM of the analog. A value of 0.50 microM for the apparent Kd was measured by the direct binding of the tritiated photoaffinity analog to tubulin. The analog is slightly more potent an inhibitor of microtubule formation than colchicine. The photoaffinity analog reacted with renal tubulin upon irradiation with a mercury lamp equipped with a 420-nm cutoff filter. Spectral and radiochemical analyses of the tubulin after photolysis and dialysis have demonstrated a stoichiometric incorporation of the photoaffinity analog in the alpha-subunit of the tubulin. Covalent labeling of tubulin with the photoaffinity analog decreases the extent of (TH)colchicine binding by more than 90%.
Research Organization:
Univ. of Texas, San Antonio
OSTI ID:
6182075
Journal Information:
J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 25; ISSN JBCHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALOIDS
ANIMALS
ANTIMITOTIC DRUGS
ANTIPYRETICS
AZIDES
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 13
CARBON ISOTOPES
CATTLE
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHEMICAL REACTIONS
CHROMATOGRAPHY
COLCHICINE
COVALENCE
DECOMPOSITION
DOMESTIC ANIMALS
DRUGS
EVEN-ODD NUCLEI
INFRARED SPECTRA
ISOTOPE APPLICATIONS
ISOTOPES
KIDNEYS
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MAGNETIC RESONANCE
MAMMALS
MICROTUBULES
NITROGEN COMPOUNDS
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
PROTEINS
REACTION KINETICS
RECEPTORS
RESONANCE
RUMINANTS
SEPARATION PROCESSES
SPECTRA
STABLE ISOTOPES
THIN-LAYER CHROMATOGRAPHY
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES