SOME PROBLEMS WITH THE DIAMINE OXIDASE (DAO) ASSAY USING PUTRESCINE AS SUBSTRATE IN RAT-LIVER

Determination of diamine oxidase (DAO) activity in rat liver preparati ons by measuring the formation of radioactive delta1-pyrroline from C- 14-putrescine is complicated by the complexity of competing metabolic pathways. This can lead to complete masking of the DAO activity presen t when rat liver homogenates are used as the enzyme source. However, s ubcellular fractionation of rat liver homogenates makes it possible to detect some putrescine oxidizing activity in the microsomal fraction when assayed at pH 8.5. When 1 mM putrescine was used as the substrate , over 90% of this activity was inhibited by 6 x 10(-4) M selegiline ( deprenyl), indicating that monoamine oxidase (MAO) rather than DAO act ivity was being measured. The observed activity was also interfered wi th by agents that reduced acetylation processes and polyamine synthesi s. A different picture appears when muM concentrations of putrescine a re used: in these conditions all interference is strongly reduced and DAO activity can be measured in rat liver microsomes. Furthermore, kin etic studies on deaminative oxidation of C-14-putrescine at concentrat ions from 1 muM to 5 mM confirm the existence of two enzymes: one with a high affinity for the substrate and similar to intestinal mucosa DA O in its sensitivity to alpha-aminoguanidine, and the other one with a low affinity and selegiline-sensitive.