The interactions between juvenile hormone (JH), lipophorin, vitellogenin, and JH esterases in two cockroach species

For the cockroach species Leucophaea maderae and Periplanata americana two major juvenile hormone (JH)-binding proteins have been identified: lipophor in (Lp) and vitellogenin (Vg). Each of these macromolecules binds JH with a n approximate affinity of K-d of 10 nM. In Leucophaea the concentration of Lp is augmented by JH during vitellogenesis at the same time when Vg is ind uced de novo. The circulating levels of each of Lp and Vg at mid-vitellogen esis are in the 10 mu M range. Similar values have been determined for Peri planeta. Total JH concentrations (bound and free) can be as high as micromo lar in Leucophaea. However, because of the large quantities of the two majo r JH-binding proteins and their high affinity for JH, we can assume that th e amount of free (unbound) JH in circulation is extremely low (the actual v alues are not know). The JH esterases (JHEs) of the hemolymph in both cockroach species have bee n isolated by anion exchange chromatography. The JHEs of Leucophaea bound t o the anion exchange resin more tightly than the JHE of Periplaneta. The V- max of the Leucophaea esterases fluctuated by a factor of 2 to 3 during vit ellogenesis. The K-m values for the two distinct esterases of Leucophaea we re similar (about 0.15x10(-6) M). On the other hand, k(cat) of the JHEs for Leucophaea at ovulation time was two to three times higher than earlier du ring vitellogenesis, i.e. 23.30 min(-1) compared to 6.20 min(-1). The JHE o f Leucophaea is shown to bind JH III with high affinity: K-d=3x10(-9) M. Ho wever, since there are only very small amounts of JH available for degradat ion (due to the binding to Lp and Vg), the quantitative removal of JH from circulation, and this includes the release of bound JH, is indeed slow, wit h a measured half-life of 6-8 h. Classical kinetic assumptions are not met in conditions where the enzyme concentrations exceed by far that of the ava ilable substrate. Nonetheless, we attempted to determine the initial veloci ty of JH hydrolysis under natural conditions, i.e. for undiluted hemolymph, by measuring the initial velocities of JH hydrolysis in serially diluted h emolymph and extrapolating to zero dilution. For in vivo conditions we esti mated an initial velocity of JH hydrolysis of <0.1 fmol mu l hemolymph(-1) min(-1), i.e. four to five orders of magnitude lower than that measured at substrate saturation in vitro. (C) 2000 Elsevier Science Ltd. All rights re served.