Purification and characterization of juvenile hormone esterase from hemolymph of the colorado potato beetle

Abstract

In the Colorado potato beetle (Leptinotarsa decemlineata), low juvenile hormone (JH) titers are necessary to initiate metamorphosis and diapause. Low JH titers coincide with high activities of JH esterase, which occur mainly in the hemolymph. The specific activity of JH esterase appeared to be highest in the last larval instar, at day 3 after the molt, and reached a value of 13.5 nmol/min/ mg. JH esterase was purified from hemolymph collected at this stage by a sequence of separation systems, including preparative nondenaturing PAGE, isoelectric focusing, and SDS-PAGE. The enzyme had a molecular weight of 120,000 and was composed of two subunits with molecular weights of 57,000, which were not linked by disulphide bridges. Isoelectric focusing revealed two forms of the enzyme with isoelectric points of 5.5 and 5.6. The Km and kcat of the purified enzyme were determined. The major form with pi 5.6 had a Km of 1.4 × 10-6M and a kcat of 0.9 s-1 and the minor form with pi 5.5 had a Km of 2.2 × 10-6M and a kcat of 1.9 s-1. The quaternary structure of L. decemlineata JH esterases, as differs from JH esterases in other species, which are monomers. © 1997 Wiley-Liss, Inc.