Ecdysteroid Secretion Research Articles

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1.1. The prothoracic gland is the main source of ecdysteroids in larvae of the cockroach Periplaneta americana.2.2. Besides ecdysone the molting gland of Periplaneta secretes 3-dehydroecdysone and proteins.3.3. The molting gland of Periplaneta is regulated in different successive steps of cooperation of nervous and neuroendocrine activity.4.4. Neurogenic effects on the molting gland via the prothoracic gland nerves are concentrated on the period of prepeak production of ecdysteroids.5.5. Prior to the 17th day, the glands secretory activity is inhibited by GABA-ergic neuronal pathways from the subesophageal ganglion.6.6. Neurogenic disinhibition by a peptidergic brain factor elicits the competence of the gland for prepeak activity, completed by the glandotropic effect of PTTH.7.7. The 17th day of the larval stage is characterized as the head critical period, i.e., after this period the ecdysteroid secretion of the gland is independent of the prothoracicotropic hormone (PTTH) from the brain.8.8. The main peak of ecdysteroid production is regulated by prothoracicotropic neuropeptids from the brain.

Daily changes in neuroendocrine control of moulting hormone secretion in the prothoracic gland of the cockroach Periplaneta americana (L.)

Time of day related changes in ecdysteroid secretion by the prothoracic gland of last instar nymphs were studied using in vitro coincubations of prothoracic glands and brains under a 12-h light:12-h dark cycle. The experiments reveal that the cells of the prothoracic gland of the cockroach nymphs do not have an endogeneous circadian oscillator determining rhythmicity of ecdysteroid secretion. PTTH release in the scotophase is responsible for the peak of ecdysteroid production during the photophase.

The Molt-Inhibiting Hormone in the American Crayfish Procambarus clarkii: Its Chemical Synthesis and Biological Activity

The molt-inhibiting hormone of the American crayfish Procambarus clarkii (Prc-MIH), a 75-residue polypeptide containing three disulfide bridges, was synthesized by chemical ligation of two peptides, i.e., synthetic Prc-MIH(1–39) and Prc-MIH(40–75)-NH2, and by subsequent folding to form the native disulfide-containing peptide molecule. The synthetic peptide was comparable to the natural Prc-MIH in inhibiting ecdysteroid secretion by in vitro bioassay and shared features with the natural Prc-MIH in some biochemical analyses. These results indicate that the chemical ligation method can be used for the synthesis of Prc-MIH. Furthermore, it was demonstrated that synthetic Prc-MIH has hyperglycemic activity, although the activity was weaker than that of the authentic crustacean hyperglycemic hormone in the American crayfish. To examine the structural requirement of the Prc-MIH for eliciting biological activity, an antibody raised against the C-terminal region (residues 55–75) and two synthetic peptides, i.e., a core region (residues 1–54) containing three disulfide bridges and the C-terminal region, were utilized. It is suggested that Prc-MIH exerts its activities through coordination between the core region and the C-terminal region.

A recombinant molt-inhibiting hormone of the kuruma prawn has a similar secondary structure to a native hormone: determination of disulfide bond arrangement and measurements of circular dichroism spectra.

In crustaceans, molt-inhibiting hormone (MIH) is presumed to regulate molting through suppressing synthesis and/or secretion of ecdysteroids by the Y-organ. Recently, a recombinant MIH of the kuruma prawn Penaeus japonicus was produced in E. coli. To approximate the secondary structure of native and recombinant MIH of P. japonicus containing six cysteine residues, the arrangements of disulfide bridges in both MIHs were determined by characterizing their enzymatic digests, and their circular dichroism spectra were measured. The arrangements of disulfide bonds in both MIHs were determined to be identical, and they were linked between Cys7 and Cys44, Cys24 and Cys40, and Cys27 and Cys53. The circular dichroism spectra of both MIHs were very close, and demonstrated that they were rich in a-helix. a-Helix contents in native and recombinant MIHs were calculated to be 49.3% and 46.0%, respectively. All these results strongly suggested that the recombinant MIH was folded in the same manner as the native MIH.

Cyclic AMP-dependent and independent stimulations of ovarian steroidogenesis by brain factors in the blowfly, Phormia regina

The involvement of cyclic-AMP (cAMP) as a potential second messenger in the neurohormonal control of ovarian steroidogenesis was investigated in the adult female blowfly Phormia regina. Individual measurements of ovarian cAMP concentrations and of ovarian biosynthesis of ecdysteroids, stimulated after a protein meal, demonstrated that steroidogenesis is preceded by a peak of cAMP in the ovaries. In vitro, ovarian steroidogenesis was stimulated by cell-permeable analogues of cAMP and by forskolin. Crude brain extracts were also able to elicit a rise of cAMP in the ovaries in vitro and the secretion of ecdysteroids into the medium: such extracts were more active before than after the protein meal, suggesting a rapid release of neuroendocrine material after feeding. Extracts were then prepared from the dorso-medial part of the brain, containing the neurosecretory cells of the pars intercerebralis (PI): these extracts were again found to stimulate the ovarian ecdysteroid secretion, but surprisingly, they failed to trigger a rise of cAMP in the ovaries in vitro. However, extracts from the rest of the cephalic nervous mass, deprived of PI, were also steroidogenic and they increased ovarian cAMP. Experiments with Rp-cAMPS, a cAMP antagonist, were not able to prevent the ecdysteroid stimulation by PI extracts, but did so partly for the extracts deprived of PI. This study thus indicates that at least two different cephalic factors are able to stimulate ovarian steroidogenesis in the blowfly, one elaborated by PI and acting via a cAMP-independent mechanism, and the other elaborated outside PI and using cAMP as a second messenger.

Neurotropic effect of juvenile hormone III in larvae of the cockroach, Periplaneta americana

Juvenile hormone III and juvenile hormone analogs (methoprene, farnesyl methyl ether) cause a short-term depression of spike activity in last instar larvae of the cockroach Periplaneta americana, specifically in the prothoracic gland nerves, both in vivo and in vitro. The decreased nervous activity in the prothoracic gland nerves leads to decreased ecdysteroid secretion by the prothoracic gland.

Interactions between Ca 2+ and cAMP in ecdysteroid secretion from the prothoracic glands of Bombyx mori

The interaction between Ca 2+ and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca 2+ from the PGs’ incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca 2+ from the medium. The cAMP agonist Sp-adenosine 3′,5′-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca 2+ from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca 2+ channel blocker verapamil. Both the Ca 2+ ionophore A23187 and the L-type Ca 2+ channel agonist S(-)•Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca 2+ and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs.

Downregulation of the cAMP signal transduction cascade in the prothoracic glands is responsible for the fenoxycarb-mediated induction of permanent 5th instar larvae in Bombyx mori

Fenoxycarb application at 48 h (day 2) of the 5th instar of Bombyx mori induced permanent larvae with prothoracic glands (PGs) exhibiting weak ecdysteroidogenic activity. Although glands from control and fenoxycarb-treated larvae exhibited similar responses to dibutyl cAMP and forskolin on day 2, forskolin could not stimulate ecdysteroid secretion from PGs of fenoxycarb-treated larvae on day 3. Glands from control larvae incubated with cholera toxin (CTX) on day 3 had increased cAMP content and enhanced ecdysteroid secretion. Cholera toxin did not stimulate ecdysteroid secretion and marginally increased cAMP content in day 3 PGs of fenoxycarb-treated larvae. After application of fenoxycarb on day 2, crude brain extracts (cBRAIN) could not increase the glandular cAMP content throughout the rest of the 5th instar of the treated larvae. Fenoxycarb did not affect the basal or cBRAIN-stimulated cAMP accumulation in control PGs on day 2 and day 3 in vitro. Application of fenoxycarb on day 2 did not affect the recombinant PTTH (rPTTH)-stimulated ecdysteroid secretion on day 3, but reduced the cBRAIN-stimulated ecdysteroid secretion on day 3 to levels similar to that of rPTTH. The combined results suggest that the cAMP signalling cascade in the PGs of B. mori becomes nonfunctional after fenoxycarb application on day 2 of the 5th instar.

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2 h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30 min incubation. On the contrary, PGs incubated with cBRAIN for 30 min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca 2+, rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca 2+ in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca 2+. Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

Localization of the mosquito insulin receptor homolog (MIR) in reproducing yellow fever mosquitoes ( Aedes aegypti)

The female mosquito takes a blood meal to produce a batch of eggs. Initiation of egg maturation and growth of oocytes is governed by several endocrine factors. Peptide factors from the brain are involved in this process and some are also responsible for the induction of ecdysone secretion. The latter appears to be required to maintain a high rate of vitellogenin synthesis. By analogy with the known functions of insulin-like molecules (e.g. bombyxins) which in insects activate the secretion of ecdysteroids, we have postulated that there is an insulin receptor homolog responsible for activation of endysone secretion in the ovary. We have recently cloned the mosquito homolog (MIR) and are now investigating its spatial and temporal distribution. Here, we have localized the insulin receptor (MIR) both at the mRNA and protein level using in situ-hybridization and immunocytochemistry. The receptor is expressed before a blood meal mainly in the nurse cells of ovaries. After a meal, follicle and nurse cells contain mRNA coding for the receptor. The intensity of expression rises in the follicle cells until they degenerate during choriogenesis. Immunocytochemical localization confirms the in situ data: the protein is present before and after a meal. Both methods confirm our previous findings by Northern blot analysis, in which the ovary was found to be the main source of the receptor mRNA. The dynamics of receptor mRNA are related to the dynamics of ecdysone secretion and its action on physiological processes.

Role of calcium in the stimulation of ecdysteroidogenesis by recombinant prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

The aim of this study was to clarify whether Ca 2+ plays a role in the mechanism by which recombinant prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by the prothoracic glands of the silkworm, Bombyx mori. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect basal ecdysteroid secretion, but greatly inhibited the stimulation of ecdysteroidogenesis by PTTH. The addition of a Ca 2+ ionophore (ionomycin or A23187) stimulated ecdysteroid secretion in a dose-dependent manner. Furthermore, thapsigargin, a potent Ca 2+-ATPase inhibitor, enhanced ecdysteroid secretion. In contrast, thimerosal, a thiol-oxidizing reagent, inhibited prothoracic gland activity. The mechanism of Ca 2+ action was examined further in relation to the cAMP effector system. Ca 2+ deprivation greatly suppressed the accumulation of cAMP in prothoracic glands treated with PTTH but had no effect on basal levels. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect the stimulation of ecdysteroid secretion in response to dibutyryl cAMP or 1-methyl-3-isobutylxanthine. The present study suggests that specific regulatory actions of Ca 2+ in prothoracic gland cells are exerted prior to cAMP generation.

Angiotensin II and Angiotensin-Converting Enzyme as Candidate Compounds Modulating the Effects of Testis Ecdysiotropin in Testes of the Gypsy Moth,Lymantria dispar1

Lymantria dispartestes synthesize immunodetectable ecdysteroidin vitroin response to the brain peptide, testis ecdysiotropin (TE), acting primarily via a cascade involving Giprotein, diacyl glycerol, and phosphokinase C. However, a component of TE activation also involves the opposite cascade, Gsprotein, cAMP, and phosphokinase A. Excess cAMP inhibits the action of TE, acting as a feedback modulator. Here, we show that bovine angiotensin II (AII) and bovine angiotensin converting enzyme (ACE) act like cAMP, inducing synthesis of immunodetectable ecdysteroid by pupal testesin vitro, but are antagonistic to coincubated TE. In addition, an insect ACE antibody clearly stains the spermatogenic cells through all stages of development, as well as testis sheath tissue where ecdysteroid is synthesized. AII induces synthesis of cAMP by pupal testesin vitro.Therefore, insect homologs of mammalian AII and ACE are good candidates for the peptides responsible for the cAMP cascade and as modulators of TE action in lepidopteran testes. Saralasin, an analog of AII that blocks angiotensin receptors in mammals, behaved like AII in inducing ecdysteroid secretion with ecdysteroidogenic effects additive to either angiotensin or ACE. Therefore, the receptors for the insect form of angiotensin on lepidopteran testis cells are probably different from those in mammals. Saralasin also inhibited ecdysteroid synthesis when combined with TE, as did AII.

Ecdysteroid release by the prothoracic gland of Gryllus bimaculatus (Ensifera: Gryllidae) during larval–adult development

The in vitro secretion of ecdysteroids from the prothoracic glands of larvae of Gryllus bimaculatus was analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (65–93%), with lesser amounts of ecdysone (7–35%). Production and release of ecdysteroids from the prothoracic glands are calcium-dependent. The rate of ecdysteroid release was low during the beginning and the end of the last two larval stages and high in between. Prothoracic glands from young adult females produced only minor amounts of ecdysteroids and ceased hormone production around day 4 after the moult.

Intracellular calcium in prothoracic glands of Manduca sexta

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP 3-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.

Ecdysteroidostatin from the house fly, Musca domestica.

Ovaries from house flies maintained on sucrose secrete large amounts of ecdysteroid when they are cultured with ovarian ecdysteroidogenic hormone, OEH. However, ovarian ecdysteroid secretion is reduced by incubation with both OEH and the ovarian ecdysteroidostatin (OES). A partially purified OES fraction from a semi-preparative reverse phase HPLC C18 column caused a 98% inhibition of ovarian ecdysteroid secretion in vitro at a concentration of 0.8 equivalents per microliter. Ovaries can be activated to produce ecdysteroid in vivo by feeding diet containing protein to flies maintained on sucrose. Ecdysteroid secretion was inhibited when the in vivo stimulated ovaries were cultured with OES. This suggests that OES does not interfere with the OEH activation mechanism, but blocks ovarian ecdysteroid synthesis or release. Furthermore, OES inhibition is reversible and ecdysteroid secretion resumes when OES is removed. Musca OES could explain the decrease in ecdysteroid levels found in flies after mid-vitellogenesis. Both adult male and female abdomens contain OES, but OES was not transferred to females during mating. Evidence is presented that OES is not a trypsin modulating oostatic factor.

Insulin-mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti.

The reproductive cycle of female mosquitoes is activated by ingestion of blood from vertebrate hosts. Shortly after feeding, neurohormones are released from the brain neurosecretory system and stimulate the ovaries to secrete ecdysteroids, which are necessary for vitellogenesis by the fat body. Because bombyxins, which are insulin-like peptides, stimulate ecdysteriodgenesis in silkworm larvae, we tested porcine insulin and found that it activates ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. To further characterize the regulation of ecdysteroidogenesis in female mosquitoes, we cloned the mosquito insulin receptor (MIR) homologue from ovarian mRNA. The sequence of the extracellular domain shows moderate homologies with vertebrate and Drosophila insulin receptor homologues, as well as with the insulin receptor-related receptor, the latter being an "orphan" receptor with an unknown function. In the intracellular domain, high homologies are observed, particularly in those subdomains that are responsible for ATP binding and kinase activity. Northern blot analysis of MIR demonstrated a highly specific expression in ovaries, and cloning experiments indicated its presence in the brain. Recombinant MIR extracts from a baculovirus expression system contained high constitutive kinase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombinant receptor was not post-translationally processed into an alpha- and beta-subunit as was expected from a putative cleavage signal. Enzymatic properties of the proreceptor are presented: the Km for ATP was between 15 and 50 microM in the presence of a synthetic substrate: maximal kinase activity to 100-fold over basic activity was reached in the presence of 1 mM manganese. Stimulation of key oogenic processes by porcine insulin and identification of a MIR indicate that insulin-like neurohormones may have an important regulatory role in mosquito oogenesis.

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh flyNeobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.

Ommochrome pigmentation of thelinea androsa seasonal forms ofPrecis coenia (Lepidoptera: Nymphalidae)

Precis coenia exhibits a seasonal color polyphenism. Animals reared under long-day conditions develop a pale tan pigmentation on their ventral hind wing (the linea form), whereas animals reared under short-day conditions develop a dark reddish-brown pigmentation (the rosa form). This developmental switch is mediated by a shift in the timing of ecdysteroid secretion at the outset of adult development. In addition, a single recessive gene mimics the effect of short-day conditions and, when homozygous, produces a constitutive rosa phenotype. This article analyzes the pigment composition of the ventral hind wing pattern of the linea and rosa forms. It presents a new thin layer chromatography system that separates ommochromes with high resolution. The linea pigmentation was found to be due to xanthommatin, while the rosa pigmentation is due to a mixture dihydro-xanthommatin and ommatin-D. Induction of the rosa form thus requires the synthesis of a new pigment (ommatin-D is a sulfate of dihydro-xanthommatin) and the stabilization of the reduced form of xanthommatin. Arch. Insect Biochem. Physiol. 36:215–222, 1997. © 1997 Wiley-Liss, Inc.

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria sarcophaga bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.

Purification and characterisation of haemolymph 3-dehydroecdysone 3 beta-reductase in relation to ecdysteroid biosynthesis in the cotton leafworm Spodoptera littoralis.

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC-RIA. The primary product was identified as 3-dehydroecdysone (approximately 82%), with lesser amounts of ecdysone (approximately 18%). Interconversion of ecdysone and 3-dehydroecdysone by prothoracic glands was not detectable. 3-Dehydroecdysone 3 beta-reductase activity was demonstrated in the haemolymph. Ecdysone, the endproduct, was characterised by reverse-phase and adsorption HPLC, chemical transformation into ecdysone 2, 3-acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and Km values for NADPH and NADH were determined to be 0.94 microM, and 22.8 microM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3-dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (> 5 microM). The results suggest that the 3-dehydroecdysone 3 beta-reductase has a high-affinity (low Km) binding site for 3-dehydroecdysone substrate, together with a lower-affinity inhibition site. The 3 beta-reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono-Q, phenyl Superose (twice), and hydroxyapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel-filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.