722 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
0–6 min 20 B, 6–46 min 20–36 B linear gradient, 0.4 B per min that means 0.3 CH
3
CN per min, 46– 52 min 36–100 B, 52–57 min 100 B, 57–65 min
100–20 B; flow rate: 100 µ
lmin. The sample from the second HPLC run was reduced to a volume of ca. 10
µ l, filled up with 20 B to an injection volume of 100
µ l and loaded onto the column. Peaks were collected and
tested for allatoregulating activity 50 brain equivalent per assay. The most significant peak eluting between
26.6 and 27.4 min showed allatotropic activity and was pure enough to be analysed by mass spectrometry and
Edman degradation.
2.5. Mass spectrometry and Edman degradation For mass spectrometry and Edman degradation 1
µ l
of the purified sample was dried down and dissolved in 3
µ l waterCH
3
CN 11, vv. A 1 µ
l sample was ana- lysed by mass spectroscopy using a MALDI-TOF instru-
ment Voyager DE-STR, PerSeptive Biosystems, Fram- ingham, MA, USA. Peptide samples were prepared
using dihydroxybenzoic acid as a matrix. For external molecular weight calibration a mixture of four synthetic
peptides was applied.
Edman sequencing was performed using the auto- mated protein sequencer 494 cLC from ABI-Perkin-
Ellmer Perkin Ellmer Biosystems, Warrington, UK. The Fast A software program was used to search for
sequence similarities in SwissProt and PIR protein data- bases.
2.6. Coelution of synthetic and native peptides Synthetic Mas-AT was purchased from Bachem
Bubendorf, Switzerland and coeluted with native pep- tide using the micro HPLC system as in the third HPLC
purification step, with the following modifications and chromatographic conditions: 10
µ l sample loop; column:
YMC-Pack ODS-AQ, 120 A ˚ , 5
µ m, 150
× 0.5 mm; sol-
vent A: 0.113 TFA in 5 CH
3
CN, solvent B: 0.1 TFA in 80 CH
3
CN; gradient: 0–6 min 20 B, 6–46 min 20–36 B linear gradient, 0.4 B per min that
means 0.3 CH
3
CN per min, 46–52 min 36–100 B, 52–57 min 100 B, 57–65 min 100–20 B; flow rate:
10 µ
lmin. After initial runs with synthetic Mas-AT and native peptide separately, both peptides were coinjected
in equal amounts of about 20 pmol.
3. Results
3.1. Purification and identification of the allatotropic peptide
In the first HPLC step bioactive material eluted between 54.5 and 55.3 min Fig. 1. This fraction caused
an eightfold increase in JH biosynthesis 30 brain equiv- alents per assay and, therefore, was selected for further
purification. Allatotropic material from the second HPLC run 53.0–54.8 min, not shown eluted from the
third HPLC-system as a single peak at around 26.6 min Fig. 2 and was the only fraction to induce a distinct
allatotropic response 50 brain equivalents per assay. The total yield of allatotropic material was estimated at
about 70 pmol, which corresponded to an amount of ca. 16 fmolbrain. The sample was subjected to sequence
analysis and mass spectrometry. Edman sequencing indi- cated an amino acid sequence GFKNDVEMMxARGF
which is similar to Mas-AT. MALDI-TOF analysis showed a mass of 1486.11 Da M
+ H
+
which corre- sponded to the theoretical value of 1486.79 Da M
+ H
+
for Mas-AT. Thus, the uncertain amino acids on pos- itions 4 and 9 could be designated as N and T, respect-
ively, and the peptide was tentatively identified as Mas- AT
with an
amino acid
sequence GFKNVEMMTARGF–NH
2
. For
additional confir-
mation of the primary structure, synthetic Mas-AT and native peptide were coinjected onto a YMC-Pack HPLC
column in equal amounts of ca. 20 pmol. A single peak eluted at 28.05 min not shown, which had almost a
double peak area ca. 200,000 units compared to the single peak areas from previous runs of both peptides
alone each ca. 110,000 units.
3.2. Effects of synthetic Mas-AT and Mas-AS on JH release in vitro
After a first incubation in medium without peptides, CC–CA-complexes were transfered into a medium con-
taining synthetic Mas-AT at concentrations ranging from 10
210
to 10
25
M Fig. 2. Mas-AT affected the CC–CA- complexes in a dose-dependent allatotropic manner.
Increasing allatotropic effects were achieved at concen- trations of 10
28
M and higher. Mas-AT caused a seven- to eightfold rise in JH release at a concentration of 10
25
M. That means an increase in JH release from about 3 to 22 pmol h
21
animal
21
. Mas-AT promoted the JH pro- duction in all ages of adult females examined day 0–11
after emergence, but maximal stimulation was obtained during the first 4 days and especially with CC–CA-com-
plexes from 2-day-old females Fig. 3, AT. Synthetic Mas-AS showed no significant effect on JH release in
the adult stage Fig. 3, AS. When CC–CA-complexes were incubated in medium containing a combination of
both peptides, Mas-AT and Mas-AS, only a slight increase in rates of JH release was observed Fig. 3,
ATAS. This indicates that the stimulating effect of Mas-AT was reduced or even abolished by the addition
of Mas-AS. The ability of Mas-AS to inhibit the allato- tropic effect of Mas-AT was dose-dependent Fig. 4.
The CC–CA-complexes incubated with 10
26
M Mas-AT produced up to seven times more JH than controls with-
723 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
Fig. 1. Chromatographic steps of S. frugiperda allatotropin isolation from 5800 brains of adult females. A Chromatogram of the first HPLC
step. B Chromatogram of the third last HPLC step. For details see Section 2. Peaks were collected from the HPLC runs and their allatoregulating activity measured with the rapid partition assay with CC–CA-complexes from 2-day-old females at a concentration of 30 brain equivalents per
assay A, lower graph and 50 brain equivalents per assay B, lower graph, respectively. Mean values of five determinations. Mas-AT: peak identified as M. sexta allatotropin.
Fig. 2. Dose response for stimulation of CC–CA-complexes from 2-
day-old females of S. frugiperda by Mas-AT. Mean values ±
SE of 10 determinations. Rate of JH release by untreated glands was ca. 3 pmol
h
21
animal
21
.
out Mas-AT. The basal rate was ca. 3 pmol h
21
animal
21
and the stimulated rate was about 21 pmol h
21
animal
21
. Addition of Mas-AS at concentrations of 10
25
and 10
26
M significantly reduced the rates of JH release. This sug- gests that Mas-AS strongly suppressed the allatotropic
effect of Mas-AT. This inhibiting effect was reduced at 10
27
M Mas-AS concentration, and disappeared altog- ether at 10
28
M concentration. In another experiment, three successive 2 h incubations were carried out and
either a single peptide or two peptides were added to the medium, according to the figure legend Fig. 5. Basal
rates of JH release were relatively constant during three consecutive 2 h incubation periods with ca. 4 pmol h
21
animal
21
Fig. 5, first group. The second group con- firmed earlier results that Mas-AS does not affect JH
release of CA from adult females of S. frugiperda. Mas- AT acted as a stimulant, and the glands returned within
2 h to unstimulated values in medium without Mas-AT Fig. 5, third group. When Mas-AS was added together
with Mas-AT, JH release rates were less than half of the hormone production rates obtained with Mas-AT alone
Fig. 5, last three groups. The inhibiting effect of Mas- AS on allatotropin activated glands was reversible Fig.
5, sixth group.
3.3. Effects of synthetic peptides of the FGL-amide peptide family on JH synthesis
In a first experiment, members of the FGL-amide alla- tostatin family which had been isolated from Helico-
verpa armigera Duve et al., 1997, and an allatostatin of the W
2
W
9
-peptide family isolated from the cricket G. bimaculatus Lorenz et al., 1995a,b, were tested for
their ability to inhibit JH release in CC–CA-complexes from 2-day-old females of S. frugiperda. None of these
724 U. Oeh et al. Insect Biochemistry and Molecular Biology 30 2000 719–727
Fig. 3. Effects of Mas-AT and Mas-AS on JH release in CC–CA-
complexes from females of S. frugiperda at the first 11 days after emergence. Mean values of five determinations. Peptides were added
during a second 2 h incubation at a concentration of 10
26
M. Mas-AT AT, Mas-AS AS, Mas-AT plus Mas-AS ATAS.
Fig. 4. Dose response for the inhibition of the allatotropic effect of
Mas-AT by Mas-AS in CC–CA-complexes from 2-day-old females of S. frugiperda. Mean values
± SE of five determinations. Rate of JH
release by untreated glands was ca. 3 pmol h
21
animal
21
. Peptides were added during the second 2 h incubation. Mas-AT AT, Mas-AT
plus Mas-AS ATAS. Negative log peptide concentrations [M] are given in parentheses.
peptides significantly inhibited JH release in vitro not shown. In a second experiment, the same peptides were
used for measuring a possible allatostatic effect on CC– CA-complexes which had been activated by Mas-AT
Table 1. Again, however, none of these peptides sig- nificantly reduced JH release during the second 2 h incu-
bation period. Of all known allatostatins, only Mas-AS is able to inhibit JH release in allatotropin activated CC–
CA-complexes of S. frugiperda.
4. Discussion
