Cloning, partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis.
cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.
Keiser
Katherine C L
KC
Heska Corporation, Fort Collins, Colorado 80525, USA.
Brandt
Kevin S
KS
Silver
Gary M
GM
Wisnewski
Nancy
N
D000818:Animals; D000906:Antibodies; D003001:Cloning, Molecular / Q000379:methods; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D005423:Siphonaptera / Q000235:genetics / Q000254:growth & development*
D000906:Antibodies; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; C055698:juvenile hormone epoxide hydrolase