Photoactivated linkage of catechol O-methyltransferase and S-adenosyl-L-methionine

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Abstract
The formation of a stably linked complex of tritiated S-adenosyl-L-methionine (AdoMet) and catechol O-methyltransferase (COMT) has been achieved by irradiating the enzyme and ligand in Tris-HCl buffer (pH 7.5) with ultraviolet light at 254 nm. The reaction is specific as shown by a number of criteria. COMT inhibitors such as S-adenosylhomocysteine can block this photoactivated linkage. The [3H]AdoMet-COMT adduct has been shown to be a homogeneous protein by Sephadex gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focussing. After extensive proteolysis of the [3H]AdoMet-COMT adduct with pronase P, one major labelled product was released. This fragment could be separated by paper chromatography and was shown to be chromatographically identical to that released from the [3H]AdoMet-phenylethanolamine N-methyltransferase adduct.

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