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.
Venkatesh
K
K
Department of Entomology, North Carolina State University, Raleigh 27695.
Abdel-Aal
Y A
YA
Armstrong
F B
FB
Roe
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006458:Hemolymph / Q000201:enzymology; D007526:Isoelectric Point; D007605:Juvenile Hormones / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D011233:Precipitin Tests
D007605:Juvenile Hormones; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase