Interaction of a fluorescent analog of N-deacetyl-N-methyl-colchicine (colcemid) with liver alcohol dehydrogenase.
The evidence for specific binding of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)--colcemid (NBD-colcemid), a fluorescent analog of colcemid (N-deacetyl-N-methyl-colchicine), to liver alcohol dehydrogenase is presented. Alcohol dehydrogenase bound NBD-colcemid in a time-dependent manner, enhanced the fluorescence intensity, and caused a large blue shift of the emission maximum of the free drug. The specificity of binding was determined for both the colchicine nucleus and the NBD moiety. The binding was not affected by the presence of alcohol or NAD in the reaction mixture. Preincubation of horse liver alcohol dehydrogenase with colcemid inhibited the binding to a considerable extent. NBD-colcemid inhibited the enzymic activity of alcohol dehydrogenase in a mixed-type noncompetitive mode with a Ki value of 32 microM, whereas colcemid showed noncompetitive inhibition with a Ki of 100 microM. The association rate constant of NBD-colcemid binding with liver alcohol dehydrogenase was 587 M-1 s-1 at 25 degrees C. The stoichiometry and dissociation constant of the binding reaction were 0.62/dimer and 12 microM, respectively. Donor quenching experiments showed that both tryptophans of alcohol dehydrogenase transferred energy to the bound NBD-colcemid. Thus, this study reports the binding of a colchicine analog to a protein other than tubulin with high affinity. It is concluded that NBD-colcemid binding to dehydrogenases is a general phenomenon, but the common structural element(s) that is responsible for the binding activity, and which exists among tubulin and dehydrogenases, has yet to be determined.
Sengupta
S
S
Department of Biochemistry, Bose Institute, Calcutta, India.
Mahapatra
P K
PK
Chakrabarti
G
G
Roy
S
S
Bhattacharyya
B
B
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002942:Circular Dichroism; D003703:Demecolcine / Q000494:pharmacology; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008099:Liver / Q000201:enzymology*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010714:Phosphatidylethanolamines; D014404:Tubulin; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D003703:Demecolcine