Affinity purification of porcine kidney enzymes that bind mercapturic acids
Mercapturic acid (N-acetylcysteine S-conjugate) formation is a major route for the metabolism and elimination of the glutathione conjugates formed from endogenous and exogenous compounds, but the enzymes of the mercapturic acid pathway are still not completely characterized. In the present study, an affinity purification strategy was developed for identifying enzymes that act on mercapturic acids. Mono- and di-mercapturates of dopamine were synthesized, purified by HPLC, and covalently coupled to an agarose column. Bound proteins were eluted with a dopamine mercapturate, trypsinized, and identified by LC-MS-MS. Identified proteins included four dipeptidase enzymes; angiotensin converting enzyme, dipeptidyl-peptidase IV, membrane dipeptidase and aminopeptidase N. These dipeptidases were tested for their role in the hydrolysis of cysteinylglycine S-conjugates in the mercapturic acid pathway. Results from this study suggest that aminopeptidase and membrane dipeptidase may be involved in the hydrolysis of cysteinylglycine S-conjugates, while angiotensin converting enzyme does not contribute to the pathway.