| Toxic levels of selenium in mammals are detoxified by reduction to hydrogen selenide and three methylation steps which require S-adenosylmethionine. The present work describes the purification and characterization of thioether methyltransferase, the enzyme which catalyzes the final step in this process. A method to assay activity was developed which measures acceptance of methyl groups from (methyl-{dollar}sp3{dollar}H) -S-adenosylmethionine by dimethyl selenide. The product, ({dollar}sp3{dollar}H) trimethylselenonium ion, is separated by HPLC and quantitated by scintillation counting. Thioether methyltransferase from mouse liver and lung resides primarily in the cytosol. In terms of specific activity (pmol/min/mg protein) the enzyme is most active in the lung (30) and liver (7). Purification from lung cytosol requires a three-step process of DEAE and gel filtration column chromatographies followed by chromatofocusing. SDS-Polyacrylamide gel electrophoresis shows a single homogenous band with a molecular mass of 28,000 daltons. Thioether methyltransferase is a single monomeric species with an isoelectric point of 5.3. Activity assays reveal a pH optimum of 6.3. The enzyme is inhibited 50% with 25 uM Sinefungin or 40 uM S-adenosylhomocysteine. Vmax and Km values for dimethyl selenide as a substrate are 15. 7 pmol/min and 0.44 uM, respectively. Our studies have also shown that this purified enzyme is capable of methylating a wide range of compounds, namely dimethyl telluride, dimethyl sulfide and many others with thioether bonds. To further test the enzyme's role in detoxification, in vivo studies were performed by injecting mice with substrate and (methyl-{dollar}sp3{dollar}H) methionine and analyzing tissue extracts and urine for (methyl-{dollar}sp3{dollar}H) sulfonium. The presence of thioether methyltransferase in mouse lung, liver, kidney and urine suggest that it functions in vivo and is physiologically relevant. Pretreatment of mice with periodate-oxidized adenosine inhibits sulfonium excretion. Antibodies have been raised in rabbits against thioether methyltransferase. The antisera was shown to be specific for thioether methyltransferase by Western blot analysis of electroblotted SDS-polyacrylamide gels and immunoprecipitation of activity. The properties of thioether methyltransferase in vitro and in vivo suggest that this newly discovered enzyme methylates seleno-, telluro- and thioethers to polar onium compounds which are more polar and more suitable for urinary excretion. |
