Characterization and temporal aspects of haemolymph juvenile hormone esterase in adult cockroach,Periplaneta americana

Daily variations in the in vitro haemolymph juvenile hormone esterase activity (hJHE) of adult male and female Periplaneta americana were monitored over a 2 week period from the time of adult emergence and throughout the first reproductive cycle of the adult female. Kinetic analysis of hJHE from females indicated an apparent K(m) JH III of 5.59+/-1.75&mgr;M (V(max)=140pmol JH III hydrolysedh(-1) per &mgr;l haemolymph). In females the mean rate of JH III metabolism in diluted haemolymph shortly after emergence was 27.5+/-1.5pmolh(-1)&mgr;l(-1) (n=16) and remained relatively low (16-32pmolh(-1)&mgr;l(-1)) over much of early adult development. Activity remained at this level during the first two days of the 4 day reproductive cycle, but showed a much increased broad peak of activity (74-93pmolh(-1)&mgr;l(-1)) at 60-72h post-extrusion. This peak lags behind the whole body JH titre peak, which could suggest that elevated levels of JH III may bring about the induction of JH esterase(s). A different pattern of JHE activity was observed in adult males. hJHE rates in males at emergence were almost twice as high (81.5+/-15.8pmolh(-1)&mgr;l(-1), n=16) as those found in females at this time, but thereafter showed a downturn to moderate levels (44-68pmolh(-1)&mgr;l(-1)) that were maintained for the remainder of the study. Rapid (FPLC) DEAE-sepharose ion exchange chromatography, ultrafiltration and fast-flow superose gel filtration chromatography were employed to achieve an efficient partial purification (166-fold) of the hJHE from cell-free plasma of reproductively active female P. americana 48-72h post-ootheca extrusion. Gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE) revealed an enzyme having apparent molecular mass of between 60 and 70kDa, whilst non-denaturing PAGE and iso-electrofocusing resolved a single acidic enzyme peak with a pI of 4.9.