Differential degradation of racemic and 10R-juvenile hormone III by cockroach ( Nauphoeta cinerea) haemolymph and the use of lipophorin for long-term culturing of corpora allata

Abstract

Degradation of racemic and 10R-juvenile hormone III was analysed in warm and cold bled haemolymph and after fractionation in a KBr gradient with two assay systems. In the partition assay, where highly diluted haemolymph or haemolymph fractions were incubated with high substrate concentrations, racemic juvenile hormone III was metabolized much more rapidly than 10R-juvenile hormone III, particularly when warm bled haemolymph was used. This suggests the existence of enzymes which preferentially degrade 10S-juvenile hormone III and their induction or activation by warming. In another assay, juvenile hormone III degradation was measured in undiluted or only slightly diluted haemolymph or fractions thereof with physiological substrate concentrations. In this assay only minor quantitative differences were seen between racemic and 10R-juvenile hormone III and both enantiomers were degraded much more efficiently in cold bled haemolymph than in warm bled haemolymph. The major metabolite formed comigrated on thin-layer chromatography with juvenile hormone III acid, but with cold bled haemolymph also highly polar metabolite(s) comigrating with juvenile hormone III acid-diol were seen. This latter metabolite was more abundant when 10R-juvenile hormone III was used as substrate. Lipophorin purified by KBr gradient centrifugation was shown to bind 10R-juvenile hormone III with a high affinity ( K d of 4.08 ± 1.0 nM). When lipophorin was added to culture medium of corpora cardiaca-corpora allata production of juvenile hormone III in vitro continued over several days; in the absence of lipophorin it dropped to very low levels after 1 day. Lipophorin was shown to keep juvenile hormone III in solution and to reduce degradation of juvenile hormone III by corpora cardiaca-corpora allata. Thus, addition of lipophorin improves corpora cardiaca-corpora allata culture conditions and allows long-term culturing.