Immunoassays of Methionine Adenosyltransferase Activity, S- Adenosylmethionine and Implications for Proliferation and Carcinogenesis

To understand how S-adenosylmethionine (SAM) concentrations fluctuate in normal and cancerous cells, particularly normal and cancerous liver cells, we have developed a novel real-time immunoassay to simultaneously measure methionine adenosyltransferase (MAT) activity and SAM levels. This method integrates the MAT-catalyzed reaction of L-Met and ATP to produce SAM and a highly sensitive immunoassay that specifically quantifies the product SAM simultaneously. Laser scanning confocal microscopy (LSCM), ELISA and flow cytometry (FCM) were used to measure SAM and S-adenosylhomocysteine (SAH) in cell lines. The cellular localization of SAM and SAH varies with cell proliferation status (i.e., SAM and SAH are mostly localized in the cytoplasm in normal cells but in the nuclei of actively proliferating cells). The levels of SAM and SAH were reduced in cancer cells, except in the early stages, when protective reactions are initiated to fight against cancer or other cellular insults. MAT-I/III was stimulated by methionine (Met<1 mM) but inhibited by S-nitrosoglutathione (GSNO, 1 mM), and the methylation index (MI) increased after Met stimulation of L02 cells. Met and GSNO inhibited MAT-II activity, and the MI decreased after Met stimulation of HepG2 cells. The highly sensitive immunoassays described here provide significant advancements and improved tools for further research involving the measurement of MAT activity, SAM and SAH under various conditions.