Partial characterization of allatinhibin, a neurohormone of Manduca sexta.

During the last larval stage, corpora allata (CA) of Manduca sexta are inactivated by a factor from the brain. Apparently the same factor (allatinhibin, AI) is secreted by day 4 Vth instar brains kept overnight in Grace's medium. AI is rapidly inactivated by heat or acid but withstands exposure to alkali and can be recovered after freezing and lyophilization. Exposure to pronase, chymotrypsin, carboxypeptidases-A and -Y, as well as leucine aminopeptidase eliminated AI activity completely, whereas after exposure to trypsin and protease XVII-S, some residual activity remained. Inactivation by pyroglutamate aminopeptidase is interpreted as being due to prolinase activity of this enzyme. Incubation of CA with gentamicin, an aminoglycoside antibiotic, affects neither their ability to produce JH in vitro nor their viability in implantation assays. However, AI did not inactivate CA in the presence of low concentrations of gentamicin. This effect was used to guard against false positive assay results possibly produced by allatotoxic contamination. AI was purified by chromatography on Sephadex G-25. All activity recovered emerged from the columns in intermediate fractions with an apparent M(r) of 1,000-2,000.

In vitro bioassay for the brain allatotropic hormone of Galleria mellonella (Lepidoptera).

An in vitro sensitive bioassay for the Galleria mellonella brain allatotropic hormone (ATTH) was developed. This assay measures the rate of juvenile hormone (JH) synthesis in corpora cardiacacorpora allata complex (CC-CA) stimulated in vitro by ATTH released from the brain during short-term in vitro incubation, or by ATTH extracted from the tissue with methanol. CC-CA of the late VIth instar (VI3) larvae were used for assessment of ATTH. The maximum activation of test CC-CA by ATTH occurred at a concentration of 2 brain equivalents (per 100 ul medium). The highest ATTH activity was exhibited by the brains of chilled VII1 larvae: ATTH extracted from freshly dissected brains, or ATTH released from these brains during 6 h in vitro incubation, activated JH synthesis in the CC-CA nearly five or four times, respectively. The brain of VII1 hydroprenetreated larvae were ATTH inactive.

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta.

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor--allatinhibin (AI)--from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 microliters medium. CA must be exposed to AI for 12-16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.