title: Apparent multiple catalytic sites involved in the ester hydrolysis of juvenile hormones by the hemolymph and by an affinity-purified esterase from Manduca sexta Johannson (Lepidoptera: Sphingidae).
abstract: The esterases which metabolize juvenile hormone (JH) in some insects may be important in regulating the hormone titer. The JH ester-hydrolyzing activity (JHE) in the larval hemolymph of the tobacco hornworm (Manduca sexta) was found to be attributed to two forms of esterase with almost equivalent activity based on selectivity and kinetics of inhibition by two 3-substituted thio-1,1,1-trifluoropropan-2-ones and a phosphoramidothioate. Neither of the two forms were inhibited by diisopropyl phosphorofluoridate or iodoacetamide. Steady-state kinetics of JH II hydrolysis supported the inhibition studies and showed that the two forms were widely different in their affinity for JH II. The activity of the hemolymph was found to be bound selectively to an affinity column synthesized by the reaction of epoxy-activated Sepharose with 3-(4'-mercaptobutylthio)-1,1,1-trifluoropropan-2-one. This column offered a quantitative, one-step purification of JH esterase with a purification factor of approximately 800 and specific activity of approximately 573 nmol JH III hydrolyzed min-1 mg protein-1. The purified protein showed only a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of approximately 65,000. However, the purified enzyme apparently revealed the same two kinetic forms as the native enzyme, which indicates that two sites of the same protein are likely to be involved in JH hydrolysis.
mesh_terms: D000096:Acetone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000378:metabolism*; D006868:Hydrolysis; D007605:Juvenile Hormones / Q000378:metabolism*; D007700:Kinetics; D007814:Larva; D007915:Lepidoptera / Q000201:enzymology*; D008433:Mathematics; D009036:Moths / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
chemical_list: D007605:Juvenile Hormones; D013438:Sulfhydryl Compounds; D000096:Acetone; C047219:3-((4'-mercapto)butylthio)-1,1,1-trifluoropropan-2-one; C047218:3-(4'-butylphenylthio)-1,1,1-trifluoropropan-2-one; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase