Purification and kinetic characterisation of juvenile hormone esterase from Drosophila melanogaster

被引:34
作者
Campbell, PM [1 ]
Oakeshott, JG [1 ]
Healy, MJ [1 ]
机构
[1] CSIRO, Div Entomol, Canberra, ACT 2601, Australia
关键词
Drosophila; juvenile hormone esterase; purification; kinetics;
D O I
10.1016/S0965-1748(98)00037-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Juvenile hormone esterase (JHE) from the prepupal stage of Drosophila melanogaster was purified about 429-fold to near homogeneity by selective precipitations, isoelectric focussing, anion exchange and gel filtration chromatography. The K-M and V-max of the purified enzyme for juvenile hormone III (JHIII) hydrolysis are 89 nM and at least 590 nmol/min/mg, respectively. JHE also hydrolyses the artificial substrate alpha-naphthyl acetate with a K-M of 120 mu M and a V-max of at least 70 mu mol/min/mg. Competition of JHIII hydrolysis by five juvenile hormones and twenty-four JH analogues showed JHE is highly selective for JHIII and JHIII bisepoxide (JHB(3)), and both may be in vivo substrates. Binding in the active site of JHE is promoted by structural features found in JHIII and JHB(3) including the epoxide groups in their natural orientations, methyl (rather than ethyl) side-chains, and the 2E,3 double bond that is conjugated with the ester group. Binding is reduced by almost any departure from these structural features of JH. Go-incubation of the haemolymph JH binding protein, lipophorin, with JHE indicates lipophorin might modulate JH hydrolysis by competition for binding of JH. (C) 1998 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:501 / 515
页数:15
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