713 M.W. Lorenz et al. Insect Biochemistry and Molecular Biology 30 2000 711–718
Table 1 Chromatographic conditions of the four HPLC runs employed for the isolation of stick insect allatostatins
HPLC run Column
Solvents
a
Gradient Flow rate mlmin
1st LiChroCART Superspher 100
A: 0.115 TFA in water 0–5 min: 0 B
1 RP-18, 100 A
˚ , 4 µ
m, B: 0.1 TFA in MeCN
5–8 min: 0–20 B 124 mm
× 4 mm with guard
8–51 min: 20–33 B linear column 4 mm
× 4 mm Merck,
gradient, 0.3 MeCN per min Darmstadt, Germany
2nd Shiseido CAPCELL PAK C
18
A: 0.13 HFBA in water 0–2 min: 5 B
1 SG 300, 300 A
˚ , 5 µ
m, B: 0.13 HFBA in MeCN
2–52 min: 5–60 B linear 250 mm
× 4.6 mm with guard
gradient, 1.1 MeCN per min column 10 mm
× 4.6 mm Grom,
Herrenberg-Kayh, Germany 3rd
Shiseido CAPCELL PAK C
8
SG A: 20 mM NH
4
Ac in water 0–40 min: 6–63 B linear
1 300, 300 A
˚ , 5 µ
m, pH 7.0
gradient, 1.14 MeCN per min 150 mm
× 4.6 mm with guard
B: 20 mM NH
4
Ac in 80 MeCN column 10 mm
× 4.6 mm Grom
4th Vydac 218TP, 300 A
˚ , 5 µ
m, A: 0.115 TFA in water
0–2 min: 5 B 0.25
250 mm ×
2.1 mm MZ B: 0.1 TFA in MeCN
2–17 min: 5–20 B linear Analysentechnik, Mainz,
gradient, 1 MeCN per min Germany
17–57 min: 20–40 B linear gradient, 0.5 MeCN per min
a
HFBA, heptafluorobutyric acid; MeCN, acetonitrile; NH
4
Ac, ammonium acetate; TFA, trifluoroacetic acid.
injected with an equal amount of the corresponding syn- thetic peptide after having run native and synthetic pep-
tides separately on the same HPLC system.
2.5. Sequence analysis The allatostatic material from the final HPLC separ-
ations was loaded onto a polybrene-coated glass-fibre filter and sequenced by automated Edman degradation
using a model 477A sequenator connected to a model 120A on-line phenylthiohydantoin analyser Applied
Biosystems, Weiterstadt, Germany.
2.6. Mass spectrometry analysis Mass spectra were aquired using a matrix-assisted
laser desorptionionisation spectrometer Bruker Reflex, Bruker Franzen, Bremen, Germany. The acceleration
voltage was set to 30 kV for the linear mode. The matrix was a saturated solution of
α -cyano-4-hydroxycinnamic
acid dissolved in waterMeCN 7:3, vv. Peptide sol- utions 0.5
µ l, ca. 1 pmol were mixed on target with the
matrix solution 1:1, vv and left to dry. Each spectrum was the average of ca. 50–200 single-shot spectra
acquired in sets of five shots.
2.7. Peptide synthesis Peptide synthesis was performed on a model 9050
peptide synthesiser Milligen, Eschborn, Germany using FmocHOBt chemistry. Peptides were synthesised in the
amide form using an Fmoc-peptide amide linker poly- ethylene
glycol–polystyrene resin
Milligen. Syn- thesised peptides were purified after cleavage from the
resin by reversed-phase HPLC and checked by mass analysis. Peptide synthesis was kindly performed by N.
Weidner Mainz.
3. Results
3.1. Peptide sequences A previous study Ga¨de et al., 1997 had shown that
all the fractions eluting between 24.6 and 54.0 min corresponding to 25.0–100 MeCN from the first
HPLC run exerted a clear allatostatic activity on cricket CA. In the present study, only the more polar fractions,
i.e., those that eluted between 25.0 and 27.8 MeCN, were chosen for further purification because these frac-
tions had shown the highest level of allatostatic activity. In total, 18 peptides were purified from these fractions
of the first HPLC run. We obtained complete structural data for nine peptides of which four are members of
the –YXFGLamide family and five belong to the W
2
W
9
amide family Table 2. The peptides identified were designated Carausius morosus allatostatins type A
and B Cam-AST A and Cam-AST B in accordance with the widely accepted nomenclature for invertebrate
neuropeptides Raina
and Ga¨de,
1988 and
the
714 M.W. Lorenz et al. Insect Biochemistry and Molecular Biology 30 2000 711–718
Table 2 Sequence, molecular mass, estimated content per brain losses during purification not taken into account; overall peptide recovery was around 60
as calculated from the peak areas and IC
50
values concentration required for 50 inhibition of JH III release by CA of 4–5 day old virgin female G. bimaculatus of the isolated peptides
Calculated M +
H
+
Calculated peptide Peptide
Amino-acid sequence for the amidated
Measured M +
H
+
content pmol per IC
50
M peptide
brain Cam-AST A1
GRQYSFGL-NH
2
927.04 927.99
0.28 0.4
× 10
28
Cam-AST A2 ADGRTYAFGL-NH
2
1070.18 n.d.
0.02 0.4
× 10
28
Cam-AST A3
a
LYDFGL-NH
2 a
927.04 n.d.
0.02 2
× 10
28
Cam-AST A4 IPMYDFGL-NH
2
955.15 970.8
b
0.17 4
× 10
28
Cam-AST A5 TSSLYSFGL-NH
2
973.08 995.6
c
0.13 1
× 10
28
Cam-AST B1 AWQDLQGGW-NH
2
1060.15 1060.5
0.78 6
× 10
28
Cam-AST B2 AWQDLNTGW-NH
2
1090.17 1090.5
2.09 5
× 10
28
Cam-AST B3 GWQDLQSGW-NH
2
1076.12 1076.9
0.73 3
× 10
28
Cam-AST B4 AWQDLQGAW-NH
2
1074.17 1074.4
0.80 3
× 10
28
Cam-AST B5
d
[AWQDLQAGW-NH
2
] 1074.17
1081.3 0.82
5 ×
10
28d
Cam-AST B6 AWQDLGSAW-NH
2
1033.13 1034.3
0.10 3
× 10
28 a
Sequence as deduced by HPLC coelution with synthetic LYDFGLamide.
b
Peptide with oxidised methionine.
c
Sodium adduct.
d
Sequence as determined by Edman degradation; the IC
50
value given here is that of the synthetic AWQDLQAGWamide.
nomenclature used for the cricket allatostatins Lorenz et al., 1995a,b; Lorenz et al., 1999a. To further confirm
the identity of the allatostatins, the synthetic peptides were run under all four chromatographic conditions used
in their isolation, and their retention times were com- pared with that of the native peptide. Furthermore, the
native peptides were co-injected onto the HPLC with their synthetic counterparts. Each pair of synthetic and
native
peptide eluted
as a
pure single
peak chromatograms not shown.
Two additional peptides Cam-AST A3 and B6 could not be completely identified by means of automated
Edman degradation sequencing and mass determination, but each could be allocated to one of the two allatostatin
families Table 2. For the peptide Cam-AST A3, Edman degradation revealed the sequence LYD but no further
amino acids were detected. Also, mass spectrometry revealed no clear-cut results. In the cockroach Blattella
germanica, the allatostatin LYDFGLamide has been iso- lated from brain extracts Belle´s et al., 1994, and from
the allatostatin precursor of the cricket G. bimaculatus, the peptide LYDFGVamide was identified unpublished
results. Therefore, it seemed to be likely that Cam-AST A3 would be identical to one of these two peptides. This
assumption was proved by comparing the retention times of the native Cam-AST 3 with synthetic LYDFGLamide
a generous gift of Dr. X. Belle´s and LYDFGVamide. Co-injection of native and synthetic peptides was not
possible since all the native peptide had been used up for Edman degradation. The native Cam-AST A3 had
the same retention times as the peptide LYDFGLamide under all four chromatographic systems used for the pep-
tide purification whereas the peptide LYDFGVamide eluted at different retention times. These results suggest
that Cam-AST A3 is identical to the cockroach peptide chromatograms not shown.
For the peptide Cam-AST B6, Edman degradation revealed the sequence AWQDLgXaw, giving faint sig-
nals for the positions 6, 8 and 9 and no definite signal for position 7. With a measured M
+ H
+
of 1034.3 for the amidated peptide, the mass of the unidentified amino
acid in position 7 was calculated to be 88 Da, indicating that it might be serine. The C-terminal sequence –SAW
has been found in the lepidopteran myoinhibiting pep- tides Mas-MIP I and II Blackburn et al., 1995. There-
fore, the peptide AWQDLGSAWamide was synthesised and, upon co-injection with the native Cam-AST B6, the
peptide eluted as a single pure peak, confirming the identity of the peptide chromatograms not shown.
For the peptide Cam-AST B5 we unequivocally obtained the amino-acid sequence AWQDLQAGW;
however, mass spectrometry revealed a M +
H
+
of 1081.3 which does not correspond to the theoretical
M +
H
+
of 1074.17.
In addition,
the synthetic
AWQDLQAGWamide did not co-elute with its native counterpart. These results indicate that this peptide may
be modified. Seven additional peptides with allatostatic activity
have been isolated from the brain extracts. However, due to the low amount of the substances and partial
impurities, we did not obtain complete sequence and mass data.
The estimated content of the isolated –YXFGLamide allatostatins ranges from 0.02 to 0.28 pmol per brain and
that of the W
2
W
9
amide allatostatins from 0.10 to 2.09 pmol per brain losses during purification not taken
into account; Table 2.
715 M.W. Lorenz et al. Insect Biochemistry and Molecular Biology 30 2000 711–718
3.2. Heterologous bioassays Synthetic peptides were tested on cricket CA in con-
centrations ranging from 10
210
to 10
26
M. All the pep- tides gave the typical sigmoid dose–response curves with
a maximum inhibition of JH III release at an allatostatin concentration of 10
-7
–10
26
M. The dose–response curves of the peptides Cam-AST A1 and B4 are shown in Fig.
1. Fifty percent inhibition of JH III release IC
50
was caused by 0.4–6
× 10
28
M allatostatin Table 2. The glands showed a complete recovery from inhibited rates
of synthesis after having been transferred to fresh medium without allatostatin not shown.
3.3. Homologous bioassays When tested on paired CA from adult egg-laying C.
morosus older than 30 days at 10
25
and 10
27
M, allato- statins of both peptide families failed to inhibit JH III
biosynthesis not shown. Glands from egg-laying C. morosus were chosen since only in those females had a
measurable JH biosynthesis been observed Lorenz et al., 1999b. The peptides Cam-AST A1, A2, B1 and B4
were also tested on CA from 20–25 day old females and on CA from penultimate larval instars activated by the
addition of 100
µ M farnesol into the incubation medium,
because only under such “stimulated” conditions we were able to measure in vitro JH biosynthesis in larval
stick insects; Lorenz et al., 1999b. Again, no inhibition of JH III biosynthesis occurred, indicating that the iso-
lated peptides do not act as allatostatins in C. morosus.
4. Discussion
