Characterization of the juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol phosphotransferase (JHDPT) from Manduca sexta Malpighian tubules

Abstract

Juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol phosphotransferase (JHDPT) were characterized from the Malpighian tubules of day 1 fifth instar Manduca sexta. An improved RP-HPLC assay is described for the major metabolites of (lOR,lIS) juvenile hormone I: diol, acid, aciddiol, and diol-phosphate. JHEH is strictly associated with membrane fractions, while JHDPT is cytosolic. JHEH may be solubilized in active form by the nonionic detergents Thesit or MEGA-8. Separation of Malpighian tubule cytosol proteins using preparative isoelectric focusing yields two zones which contain JHDPT activity, at pi 4.8-5.1 and 6.8-8.2. The partially purified JHDPT from either zone requires both ATP and Mg2+ for activity, so this enzyme may be formally called either ATP:juveni/e hormone diol phosphotransferase or juvenile hormone diol kinase (EC 2.1.7.3). Metabolites more polar than JH I aciddiol and JH I diol-phosphate are generated in vivo from either [3H]JH I or 13H]JH I diol. 0 1995 Wiley-Liss, inc.

Juvenile Hormone Diol Kinase

“…Divalent Cation Requirement-Previous studies with JHDK indicated that Mg 2ϩ and ATP are required for activity (7). This observation was reconfirmed by preincubating the enzyme with 5 mM EDTA and 2 mM EGTA and performing the standard activity assay (see "Experimental Procedures").…”

“…The samples were analyzed using a solid probe with a stepwise temperature gradient (from 35 to 150°C at 115°C/min and then from 150 to 250°C at 100°C/min). Incubating [10-3 H]JH diol (compound 3) with esterase from Tenebrio molitor pupal hemolymph yields [10-3 H]JH acid diol (JHAD; compound 8) (18). We prepared [10-3 H]JHAD phosphate (JHADP) by incubating [10-3 H]JHDP (compound 11; enzymatically prepared using purified JHDK) with JHE from T. molitor pupal hemolymph.…”

“…Our standard activity assay was developed from a basic partition assay (7). It consists of the following steps.…”

“…Two studies have been conducted using Manduca sexta tissues dissected and maintained in vitro, one with Malpighian tubules (7) and the other with fat body and imaginal discs (8). The classic model for JH metabolism in insects consists of two primary pathways of inactivation: epoxide hydrolysis to form the diol metabolites and ester cleavage to form the acid metabolites.…”

Simultaneous Preparation of Both Enantiomers of Juvenile Hormones Labeled at C-10 with Tritium at High Specific Activity

“…Acidic hydrolysis of the unnatural isomer (10S,11R)-JH I gives (10S,11S)-JH I diol, which is the predominant product of enzymatic hydrolysis of the natural (10R,11S)-JH I (while we are not aware of any proof that insect JHEH has the same stereoselectivity for JH enantiomers as the mouse cytosolic epoxide hydrolase, it is highly likely that they use the same mechanism and therefore give the same stereochemical outcome). In fact, our reason for initiating this project was to obtain pure (10S,11S)-JH I diol to use as a substrate for JH diol kinase (17), an enzyme which seems to form the principal end product metabolite of JH I metabolism, JH I diol phosphate (18). We found that use of (10R,11R)- [12, H 2 ]JH I diol prepared by acidic hydrolysis of (10R,11S)- [12, H 2 ]JH I from a batch prepared according to (2) with the modifications reported by (17) gave high backgrounds in the JH diol phosphotransferase assay compared with the more rigorously purified and natural isomer of the JH diol, (10S,11S)- [10-3 H]JH I diol, reported in this paper.…”