Insect Biochemistry and Molecular Biology 30 (2000) 529–540 www.elsevier.com/locate/ibmb Isolation of juvenile hormone esterase and its partial cDNA clone from the beetle, Tenebrio molitor Beth Ann Thomas, Andrew C. Hinton, Haim Moskowitz, Tonya F. Severson, Bruce D. Hammock * Department of Entomology, One Shields Avenue, University of California, Davis, CA 956 16, USA Received 12 July 1999; received in revised form 13 January 2000; accepted 18 January 2000 Abstract Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme has been targeted for development of biologically-based insecticides. JHE was partially purified from the beetle, Tenebrio molitor, using a transition state analog as the affinity ligand. Two forms of JHE were characterized by activity analysis, isoelectric focusing, two-dimensional SDS–PAGE and N-terminal sequence analysis. The esterase is associated with two proteins of sizes 71 and 150 kDa, both of which are active on JH III. A partial cDNA clone for the enzyme was isolated based on the sequence of N-terminal and internal peptides. Its sequence indicates that JHE from T. molitor and Heliothis virescens may have a common origin.  2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Esterase; Affinity chromatography; Juvenile hormones; Insects; Hormones; Tenebrio molitor 1. Introduction Juvenile hormones (JHs), such as JH III (methyl(2E, 6E)-[10R]-10, 11-epoxy-3, 7, 11-trimethyl-2, 6- dodecanoate) (Fig. 1), are insect hormones that are involved in the regulation of the development of insects. These hormones are also required in the adult form of many insects for the regulation of reproductive processes such as oogenesis (Sehnal, 1985). In order for metamor- phosis to occur in holometabolous insects, the JH titer must decrease during the last larval stage, resulting in pupation. JH titers must remain low during metamor- phosis of the pupa in butterflies and moths (Lepidoptera). This decrease in JH is modulated, in part, by juvenile hormone esterase (JHE), which hydrolyzes the methyl ester of JH to the corresponding carboxylic acid (Fig. 1). There is now indication that the JH acid metabolite also may have intrinsic biological activity (Ismail et al., 1998). * Corresponding author. Tel.: + 1-530-752-7519; fax: + 1-530-752- 1537. E-mail address: bdhammock@ucdavis.edu (B.D. Hammock). 0965-1748/00/$ - see front matter  2000 Published by Elsevier Science Ltd. All rights reserved. PII:S0965-1748(00)00020-5 JHE (EC 3.1.1.1), a member of the the carboxylester- ase family, has been purified and characterized from sev- eral species of insects, mainly those in the lepidopteran order including Trichoplusia ni (Abdel-Aal and Ham- mock, 1988), Heliothis virescens (Hanzlik and Ham- mock, 1987) and Manduca sexta (Jesudason et al., 1990). However, there are only a few examples of characterization of this enzyme from other insect orders, such as beetles (order Coleoptera) (Connat, 1983; Stauffer et al., 1997 in Tenebrio molitor; Vermunt et al., 1998a,b in Leptinotarsa decemlineata). The JHEs of Lepidoptera have the same kcat/km ratio on the three major forms of JH homologues (Abdel-Aal and Ham- mock, 1985; Abdel-Aal et al., 1988). Recent studies on JHE enzymes have utilized JH III as a substrate due to the fact that it is now the only commercially available JH in radiolabeled form. However, JH III is thought to be the sole JH of Coleopterans (Grieneisen et al., 1997; Baker, 1990). A Dipteran JHE from Drosophila mel- anogaster was recently characterized by Campbell et al. (1998) and is likely to be the same JHE reported by Rauschenbach et al. (1995) and Khlebodarova et al. (1996). Recently, Vermunt et al. (1998b) reported a dimeric form of JHE from L. decemlineata by native