Effect of Streptomycin on Urate Oxidase Activity

Abstract

1. Crude urate oxidase solutions prepared by alkaline extraction of acetone powder prepared from ox kidney are irreversibly inhibited by streptomycin. The inhibition by 3mM streptomycin of enzyme extracted with 0.2M ammonia was virtually total in 30 minutes at 37°. 2. The inhibition was very sensitive to pH: inhibition occurred only at higher pH levels. The ammonia-extracted enzyme was 50% inhibited at pH 10.0, while tris-extracted enzyme was 50% inhibited at pH 10.3. 3. An ammonium sulphate precipitate, prepared from an ammonia extract, was not affected by streptomycin unless all traces of ammonium sulphate were removed. This was shown to be due to depression of pH of the enzyme solution to a region where the enzyme is insensitive to streptomycin, but not due to the removal of some cofactor. 4. Studies using the chelating agents EDTA, neocuproin, and cuprizone ruled out the participation of free metal ion in the inhibition. 5. Inhibition of the ammonia-extracted enzyme was associated with precipitation of protein; that of tris-extracted enzyme was not. The precipitate contained active enzyme, so streptomycin has two independent effects on the enzyme: a) precipitation b) inactivation. 6. The pH dependence of inhibition can be accounted for by assuming that an Є-amino group of a lysine moiety is involved in the inhibition, but cannot be accounted for by assuming that the guanido groups of streptomycin are involved. The unionized form of the Є-amino group could react with the iminium form of streptomycin. 7. Guanidine inhibits urate oxidase (Truscoe, 1965), so the guanidino groups of streptomycin could be responsible for the inhibition by the latter. This view is supported by the observation of Bently (unpublished work) that the inhibition by guanidine displays a sensitivity to pH which is very similar to that of SM. 8. Precipitation of protein in the ammonia system was non-specific towards the various species of protein in the solution, and precipitation of enzyme occurred at the same rate as for the contaminating, non-enzyme, protein. Thus the mechanism of precipitation must involve some quite general feature of protein molecules. 9. 50% of the protein was precipitated at pH 9.4; precipitation increased with increase in pH up to pH 10.2, where all protein was precipitated. 10. The pH dependence of protein precipitation can be accounted for by assuming that ammonia (pKa = 9.26) reacts with streptomycin to form bis(α -hydroxystreptomycyl)-amine; this latter would crosslink between protein molecules in some way, and so cause precipitation.