Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates |
| |
Authors: | J H Hurst M L Billingsley W Lovenberg |
| |
Affiliation: | Laboratoire de Biochimie Endocrinienne, INSERM-U 244, CNRS-ERA 942, USMG, CERMO, Domaine Universitaire, 38402 Saint Martin d''Hères, France |
| |
Abstract: | Radioactivity from 3H-[methyl]-S-adenosyl-L-methionine (AdoMet) was covalently bound to protein-O-carboxylmethyltransferase and phenylethanolamine N-methyltransferase following 10-15 min irradiation by short-wave ultraviolet light. This photoaffinity binding of 3H-[methyl]-AdoMet was blocked by S-adenosylhomocysteine and sinefungin, but was not affected by 5 mM dithiothreitol. The binding was also inhibited by including methyl acceptors such as calmodulin (protein-O-carboxylmethyltransferase) or phenylethanolamine (phenylethanolamine N-methyltransferase) in the photoaffinity incubation. Staphlococcus V8 protease digests of 3H-[methyl]-AdoMet/enzyme complexes revealed that the primary structure around the AdoMet binding site is different in these two enzymes. Thus, protein-O-carboxylmethyltransferase, a large molecule methyltransferase, can covalently bind 3H-[methyl]-AdoMet in a manner similar to that of phenylethanolamine-N-methyltransferase. |
| |
Keywords: | AdoMet, S-adenosyl-L-methionine AdoHcy, S-adenosylhomocysteine HEPES, (N-2-hydroxyethylpiperazine-N-2 ethane sulfonic acid UV, ultraviolet light SDS, Sodium dodecyl sulfate PAGE, Polyacrylamide gel electrophoresis Tris, Tris (hydroxymethyl) aminomethane EDTA, Ethylenediamine tetra acetic acid PEO, Phenylethanolamine |
本文献已被 ScienceDirect 等数据库收录! |
|