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-deac etyl-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 bi nding was not affected by the presence of alcohol or NAD in the reacti on mixture. Preincubation of horse liver alcohol dehydrogenase with co lcemid inhibited the binding to a considerable extent. NBD-colcemid in hibited the enzymic activity of alcohol dehydrogenase in a mixed-type noncompetitive mode with a K-i value of 32 mu M, whereas colcemid show ed noncompetitive inhibition with a K-i of 100 mu M. The association r ate constant of NBD-colcemid binding with liver alcohol dehydrogenase was 587 M(-1) s(-1) at 25 degrees C. The stoichiometry and dissociatio n constant of the binding reaction were 0.62/dimer and 12 mu M, respec tively. Donor quenching experiments showed that both tryptophans of al cohol dehydrogenase transferred energy to the bound NBD-colcemid. Thus , this study reports the binding of a colchicine analog to a protein o ther than tubulin with high affinity. It is concluded that NBD-colcemi d binding to dehydrogenases is a general phenomenon, but the common st ructural element(s) that is responsible for the binding activity, and which exists among tubulin and dehydrogenases, has yet to be determine d.