PURIFICATION AND CHARACTERIZATION OF JUVENILE-HORMONE ESTERASE HEMOLYMPH OF THE COLORADO-POTATO-BEETLE (VOL 35, PG 261, 1997)

In the Colorado potato beetle (Leptinotarsa decemlineata), low juvenil e hormone (JH) titers are necessary to initiate metamorphosis and diap ause. Low JH titers coincide with high activities of JH esterase, whic h 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 m oult, and reached a value of 13.5 nmol/min/mg. JH esterase was purifie d from hemolymph collected at this stage by a sequence of separation s ystems including preparative nondenaturing PAGE, isoelectric focusing and SDS-PAGE. The enzyme was demonstrated to have a molecular weight o f 120,000 and was composed of two subunits with molecular weight of 57 ,000, which were not linked by disulphide bridges. Isoelectric focusin g revealed two forms of the enzyme with isoelectric points of 5.5 and 5.6. The K-m and k(cat) of the purified enzyme were determined. The ma jor 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 form with pI 5.5 had a K-m of 2.2 x 10(-6) M and a k(cat) 1.9 s(-1). The quaternary structure of L. decemlineata JH ester ase as a dimer, differs from JH esterases in other species, which are monomers.