Characterization of affinity-purified juvenile hormone esterase from the plasma of the tobacco hornworm, Manduca sexta.

Juvenile hormone (JH) esterase found primarily in the hemolymph and tissues of insects is a low abundance protein involved in the ester hydrolysis of insect juvenile hormones, JHs. The enzyme was purified from the larval plasma of wild-type Manduca sexta using an affinity column prepared by binding the ligand, 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one (MBTFP), to epoxy-activated Sepharose. The purification was greater than 700-fold with a 72% recovery, and the purified enzyme appeared as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis, reverse phase high performance liquid chromatography, and amino acid sequence analysis. The molecular weight was 66,000. The plasma JH esterase in wild-type, black, and white strains of M. sexta was similar when analyzed by immunotitration, wide range (pH 3.5-9.0) isoelectric focusing, and inhibition with MBTFP and 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP). Inhibition studies revealed a sensitive and insensitive form (I50 = 10(-9) and 10(-6) M, respectively) in these three biotypes. Narrow range isoelectric focusing (pH 4.0-7.0) indicated the presence of two major isoelectric forms with pI values of 6.0 and 5.5, but their inhibition kinetics with OTFP and O,O-diisopropyl phosphorofluoridate were identical.