PURIFICATION AND CHARACTERIZATION OF JUVENILE-HORMONE ESTERASE FROM HEMOLYMPH OF THE COLORADO POTATO BEETLE

In the Colorado potato beetle (Leptinotarsa decemlineata) low juvenile hormone (JH) titers are necessary to initiate metamorphosis and diapa use. Low JH titers coincide with high activities of JH esterase, which occur mainly in the hemolymph. The specific activity of JH esterase a ppeared lo be highest in the last larval instar, at day 3 after the mo lt, 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 sys tems, including preparative nondenaturing PAGE, isoelectric focusing, and SDS-PACE. The enzyme had a molecular weight of 120,000 and was com posed 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 K-m and k(c at) of the purified enzyme were determined. The major form with pI 5.6 had a K-m of 1.4 x 10(-6)M and a k(cat) of 0.9 s(-1) and the minor fo rm with pI 5.5 had a K-m of 2.2 x 10(-6)M and a k(cat) of 1.9 s(-1). T he quaternary structure of L. decemlineata JH esterases, as differs fr om JH esterases in other species, which are monomers. (C) 1997 Wiley-L iss, Inc.