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S-adenosyl-L-methionine:farnesoic acid O-methyltransferase of Passalus cornutus: purification and characterization
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|dc.description.abstract||In Coleoptera, the final steps in juvenile hormone (JH) biosynthesis are believed to be methylation of farnesoic acid (FA) followed by epoxidation. Source of the methyl group is S-adenosyl-L-methionine, and the reaction is catalyzed by S-adenosyl-L-methionine:farnesoic acid 0-methyltransferase (FAMT). This enzyme was purified to homogeneity from a cytosolic preparation of Passalus cornutus corpora allata. The molecular weight of the purified enzyme was determined to be 28,100 by SDS-polyacrylamide gel electrophoresis. Three isoforms (pI 5.2, 4.8, 4.6) of FAMT were observed by chromatofocusing. The purified enzyme was photoaffinity labeled with S-[methyl3H]adenosyl-L-methionine. only a single polypeptide band with an electrophoretic mobility identical to that of FAMT was radiolabeled. Enzyme kinetics studies revealed that the Km for S-adenosyl-L-methionine is 5.3 x 104 M and that the Km for FA is 2.1 X .10-7 M. The substrate specificity of FAMT was tested with a competition enzyme assay. Purified FAMT was assayed in the presence of equimolar amounts of FA, JH-I acid, JH-II acid, and JHIII acid, with S-[methyl-3H]adenosyl-L-methionine as the radiolabeled substrate. The resulting methyl esters were analyzed by HPLC. The ratio of products (methyl farnesoate : JH-I : JH-II : JH-III = I : 0.14 : 0.05 : 0.02) shows that the enzyme has highest affinity for FA and suggests that FA is indeed its natural substrate. The result also supports the presumed sequence for JH biosynthesis (methylation followed by epoxidation) in CA of Coleoptera.||en|
|dc.publisher||Texas A&M University|
|dc.rights||This thesis was part of a retrospective digitization project authorized by the Texas A&M University Libraries in 2008. Copyright remains vested with the author(s). It is the user's responsibility to secure permission from the copyright holder(s) for re-use of the work beyond the provision of Fair Use.||en|
|dc.title||S-adenosyl-L-methionine:farnesoic acid O-methyltransferase of Passalus cornutus: purification and characterization||en|
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