CHARACTERIZATION OF THE JUVENILE-HORMONE EPOXIDE HYDROLASE (JHEH) ANDJUVENILE-HORMONE DIOL PHOSPHOTRANSFERASE (JHDPT) FROM MANDUCA-SEXTA MALPIGHIAN TUBULES

Juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol ph osphotransferase (JHDPT) were characterized from the Malpighian tubule s of day 1 fifth instar Manduca sexta. An improved RP-HPLC assay is de scribed for the major metabolites of (10R, 11S) juvenile hormone I: di ol, acid, acid-diol, and diol-phosphate. JHEH is strictly associated w ith membrane fractions, while JHDPT is cytosolic. JHEH may be solubili zed in active form by the nonionic detergents Thesit or MEGA-8. Separa tion of Malpighian tubule cytosol proteins using preparative isoelectr ic focusing yields two zones which contain JHDPT activity, at pl 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:juvenile hormone diol phosphotransferase or juvenile hormo ne diol kinase (EC 2.1.7.3). Metabolites more polar than JH I acid-dio l and JH I diol-phosphate are generated in vivo from either [H-3]JH I or [H-3]JH I diol. (C) 1995 Wiley-Liss, Inc.