Abstract
Juvenile hormone epoxide hydrolase (JHEH), which may play a pivotal role in regulating insect juvenile hormone (JH) titer along with JH esterase, was identified in tobacco hornworm (Manduca sexta) eggs by using photoaffinity analogs of JHs. The UV light-induced covalent labeling with [3H]epoxyhomofarnesyl diazoacetate, a JHII analog, revealed a membrane-associated 50-kDa protein that was selectively and specifically labeled. This 50-kDa protein was copurified 171-fold with the JHEH activity to homogeneity through DEAE-Sephacel, Mono Q, and hydroxylapatite columns, which led us to conclude that the labeled 50-kDa protein was a JHEH. The steady-state kinetics of the purified microsomal JHEH showed that it followed Michaelis-Menten kinetics with Km values of 0.61, 0.55, and 0.28 microM for JHI, II, and III, respectively, and that JHIII showed a significantly higher Vmax than JHI or JHII. JH acid was also converted to the corresponding diol at a rate 4-fold slower than the corresponding JH. Thus, the differences in the binding of substrate and the rate of turnover by JHEH were affected by the epoxyfarnesoate ester moiety of JH and the difference between the cis-11-methyl group of JHIII versus the cis-11-ethyl group of JHI and II. Purified JHEH showed optimal enzyme activity at pH 7.5-8.5. Interestingly, the presence of recombinant M. sexta JH binding protein (JHBP) dramatically decreased the degradation of JH by JHEH in vitro. Since the cytosolic JHBP in eggs closely resembles the hemolymph JHBP, we suggest that cytosolic JHBP may play a role in protecting JHs from JHEH in vivo. Furthermore, JHEH may play a significant role in the secondary metabolism of JH acid generated by JH esterase.
Highlights
Since the cytosolicJHBP ineggs closely resembles the hemolymph J H bindingprotein (JHBP), we suggest that cytosolicJHBP may play a role in protecting juvenile hormone (JH) from Juvenile hormone epoxide hydrolase (JHEH) invivo
Since the purified JHEH concentrated using a Centricon-30 was in 100 mM potassium phosphate buffer, pH 7.5, the pH value of the mixture was measured after adding the buffer solution
The developmental profile demonstrated that labeled 50-kDa protein existed in all embryonic stages as well as in all ovarian stages and in the crushed first instarwith no significant differences in the labeling.' These labeled proteins (32 and 50 kDa) were found in embryos isolated from the eggs, confirming that these were embryonic proteins
Summary
PHOTOAFFINITY LABELING, PURIFICATION, AND CHARACTERIZATION FROM TOBACCO HORNWORM EGGS*. Juvenile hormone epoxide hydrolase (JHEH),which may play a pivotal role in regulating insect juvenile hormone (JH) titeralong with J H esterase, was identified in tobacco hornworm (Manduca sexta) eggs by using photoaffinity analogs of JHs. The U V light-induced covalent labeling with [SH]epoxyhomofarnesyl diazoacetate, a JHII analog, revealed a membraneassociated SO-kDa protein that was selectively and labeled. JHEH metabolizes JHIII 6.6 times faster than JHI in the egg homogenate of M. sexta (Share et al, esterase; JHEH, JHepoxide hydrolase; JHBP, JHbinding protein; CMC, critical micelle concentration;CHAPS, 3-[(3-cholamidepropyI)dimethylammonio]-l-propanesulfonate; PMSF, phenylmethylsulfonyl fluoride; EHDA, epoxyhomofarnesyl diazoacetate; EBDA, epoxybishomofarnesyldiazoacetate; FPLC, fast proteinliquid chromotography; BSA, bovine serum albumin; EtOAc, ethyl acetate; TLC, thin layer chromatography. Since the purified JHEH concentrated using a Centricon-30 was in 100 mM potassium phosphate buffer, pH 7.5, the pH value of the mixture was measured after adding the buffer solution. In order to denature the JH-JHBP complex, the reaction mixture was incubated at 90 "C for 1 min in the presence of 10 mM dithiothreitol
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