MECHANISM OF THE EFFECT OF ORGANIC-SOLVENTS AND OTHER PROTEIN DENATURANTS ON NEOCARZINOSTATIN ACTIVITY

Several alcohols and other organic solvents enhance the in vitro DNA cutting activity of neocarzinostatin (NCS). Hexamethylphosphoramide is the most potent of the stimulators tested. Kinetic analysis of the DNA scission by NCS in the presence of 2-propanol shows that the solvent effect is on the Vmax and not on the Km. All the solvents, above their optimal levels, inhibit NCS activity, and they also increase the rate of its inactivation on preincubation in the absence of deoxyribonucleic acid (DNA). Among the other protein denaturants, urea stimulates the activity of NCS over a wide range of concentrations. In contrast, guanidine hydrochloride at subdenaturing levels strongly inhibits the reaction. One possible explanation for the stimulation of NCS activity by organic solvents and urea is that by breaking down water-cooperative structures they allow increased motility of the antibiotic to a conformation favorable for reduction of its disulfide bonds to generate an active species of the drug for its interaction with DNA. The possible involvement of the nonprotein chromophore of NCS in the activation process is also discussed. In addition, NCS rendered inactive by various treatments has a lower isoelectric point and specifically blocks the activity of native NCS. These results indicate that there are a limited number of DNA binding sites for which the two forms of NCS compete and provide further evidence that direct interaction between drug and DNA is responsible for its biological effects. Since several of the treatments generating the inhibitory species of NCS also cause chromophore release and since auromomycin-induced scission of DNA is blocked by its nonchromophore-containing form (macromomycin), the possibility is raised that loss of chromophore from the protein generates the inhibitory species. © 1979, American Chemical Society. All rights reserved.