The C-terminus shortened analogs of the insect peptide oostatic hormone with accelerated acitivity

Article abstract of DOI:10.1006/bioo.1998.1092

Shortened analogs 1a-1f of the oostatic hormone 1g with a decreased number or an omission of the C-terminal Pro residues were synthesized for investigation of their effect on oogenesis in the flesh fly Sarcophaga bullata Parker (Diptera). Peptides 1b-1g were prepared by solid-phase synthesis on polystyrene/divinylbenzene polymer with a 2-chlorotrityl linker using Fmoc/tBu strategy in dimethylformamide (DMF). Tetrapeptide 1a, devoid of all the carboxy-terminal Pro residues, was prepared by stepwise procedure in DMF solution from HCl-H-Ala-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu, and Boc-Tyr-OSu. In general, the peptides assayed affected processes of egg development in 20-80% of ovarioles causing changes in the follicular epithelium, proliferation of its nuclei, and cell division toward the inner part of the egg chamber. This process leads to the occurrence of a multilayered follicular epithelium which later fills up almost the whole egg chamber and then degenerates. Such ovarioles are never able to produce eggs. The shortening of the peptide sequence accelerated the observed effects on egg development. The application of the ¹²⁵I-labeled peptides made possible to express quantitatively the time dependence of the radioactivity distribution in the insect body, including ovaries through which it is transferred to the next F1 generation. The radioactivity was measured in larvaes and, consequently, in newborn flies. For different peptide sequences applied, different radioactivity distributions in the insect body were found using the same radiolabeling.

Full text of DOI:10.1006/bioo.1998.1092

BIOORGANIC CHEMISTRY 26, 131–140 (1998)
ARTICLE NO. BH981092
The C-Terminus Shortened Analogs of the Insect Peptide
Oostatic Hormone with Accelerated Activity
Jan Hlavacek,* Richard Tykva,* Blanka Bennettova,† and Tomislav Barth*
´ ˇ
´
*Institute of Organic Chemistry and Biochemistry, Academy of Sciences,
166 10 Prague, Czech Republic; and †Institute of Entomology, Academy of Sciences,
ˇ
370 05 Ceske´ Budeˇjovice, Czech Republic
Received February 11, 1998
Shortened analogs 1a–1f of the oostatic hormone 1g with a decreased number or an omission
of the C-terminal Pro residues were synthesized for investigation of their effect on oogenesis
in the flesh fly Sarcophaga bullata Parker (Diptera). Peptides 1b–1g were prepared by solid-
phase synthesis on polystyrene/divinylbenzene polymer with a 2-chlorotrityl linker using
Fmoc/tBu strategy in dimethylformamide (DMF). Tetrapeptide 1a, devoid of all the carboxy-
terminal Pro residues, was prepared by stepwise procedure in DMF solution from HCl-H-
Ala-OMe using Boc-Pro-OH, Z-Asp(OtBu)-OSu, and Boc-Tyr-OSu. In general, the peptides
assayed affected processes of egg development in 20–80% of ovarioles causing changes in the
follicular epithelium, proliferation of its nuclei, and cell division toward the inner part of the
egg chamber. This process leads to the occurrence of a multilayered follicular epithelium
which later fills up almost the whole egg chamber and then degenerates. Such ovarioles are
never able to produce eggs. The shortening of the peptide sequence accelerated the observed
effects on egg development. The application of the 125I-labeled peptides made possible to
express quantitatively the time dependence of the radioactivity distribution in the insect body,
including ovaries through which it is transferred to the next F1 generation. The radioactivity
was measured in larvaes and, consequently, in newborn flies. For different peptide sequences
applied, different radioactivity distributions in the insect body were found using the same
radiolabeling.
1998 Academic Press
INTRODUCTION
Oostatic or antigonadotropic insect hormones have been found in a number of
insect species (1). Some time ago, Borovsky et al. isolated another member of this
class of reproductive and developmental hormones from mosquito and described
it (2) to be an unusual decapeptide 1g containing six C-terminal Pro (3) residues.
This molecule was postulated to affect oogenesis through modulation of ovarian
ecdysteroid synthesis, gut trypsin synthesis, or egg development neurosecretory
hormone release in the Diptera and to influence a juvenile hormone action on
ovarian yolk uptake in some Hemiptera. The peptide was designed to function as
a signal that terminates vitellogenesis.
In our preliminary communication (4) we described the solution synthesis and
oostatic properties of some peptides with the sequence of the oostatic hormone 1g
and its shorter analogs 1d–1f, differing in a number of the C-terminal Pro residues.
131
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

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HLAVACEK ET AL.
132
A comparative study of the hormone 1g and one of the shorter analogs (1e) revealed
certain effects on processes of egg development. They were manifested by distur-
bances of yolk deposition, changes of follicular epithelium, and formation of nonvia-
ble eggs. The presence of Tyr in the peptides prepared allowed us to carry out the
radiotracer distribution studies using 125I-labeled molecules of 1d and 1g and we
quantitatively described a presence of the labeled peptide material in various parts
of the insect body (ovaries, head, intestine).
H-Tyr-Asp-Pro-Ala-Pron-OH
1a n ϭ 0, 1b n ϭ 1, 1c n ϭ 2, 1d n ϭ 3, 1e n ϭ 4, 1f n ϭ 5, 1g n ϭ 6
2a Boc-Pro-Ala-OMe
2b Z-Asp(OtBu)-Pro-Ala-OMe
2c Boc-Tyr-Asp(OtBu)-Pro-Ala-OMe
2d Boc-Tyr-Asp(OtBu)-Pro-Ala-OH
The results obtained in our first study have inspired us with an idea to perform
similar studies on analogs with a peptide sequence even more shortened from the
C-terminus. Therefore, we synthesized peptides 1a–1c to compare their oostatic
properties with peptides described earlier, including the native decapeptide 1g.
Tetrapeptide 1a, devoid of all C-terminal Pro residues, was prepared by stepwise
procedure in solution. The synthesis was started from HCl и H-Ala-OMe which
was coupled to Boc1-Pro-OH in DMF using DCC and HOBt in the presence of
DIEA. The protected dipeptide 2a was treated with TFA and the resulting TFA.H-
Pro-Ala-OMe acylated by Z-Asp(OtBu)-OSu in DMF to give the protected tripep-
tide 2b. After removal of the Z-protecting group by hydrogenolysis on Pd black in
MeOH, the Boc-Tyr-OSu was used to complete the sequence of the protected
tetrapeptide 2c which was saponified to acid 2d which was finally deprotected by
the mixture TFA–anisole (9 : 1).
Peptides 1b and 1c and the larger peptides described previously (1d–1g, Ref. 4)
were prepared by solid-phase approach using a polymer with a 2-chlorotritylchloride
linker. We have utilized Fmoc/tBu protection of amino acids which were coupled
in DMF by means of DCC/HOBt. The Fmoc protecting group has been removed
by piperidine in DMF.
In the last step of the syntheses the peptides were deprotected and split off the
resin by trifluoroacetic acid in the presence of anisole.
The peptides synthesized were purified by preparative HPLC and characterized
by means of amino acid analysis, mass spectrometry, and analytical HPLC.
To illustrate the fate of the shortened analogs after their application to insect,
basic morphological, histological, and radiotracer studies were carried out using
tetrapeptide 1a and pentapeptide 1b, respectively, including their 125I labeling. The
results were compared with those obtained for the larger peptides.
1 Abbreviations used: Boc, tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide; DIEA, diisopro-
pylethylamine; DMF, dimethylformamide; HOBt, hydroxybenzotriazole; OSu, succinimide ester; tBu,
tert-butyl; TDM, 4,4Ј-tetramethyldiamino-diphenylmethane; TFA, trifluoroacetic acid.

ACCELERATED ACTIVITY OOSTATIC HORMONE ANALOGS
133
MATERIAL AND METHODS
Materials
Z-Asp(OtBu)-OSu was purchased from Bachem (Switzerland), whereas all other
amino acid derivatives were prepared in our laboratory following general protocols
(5) and were checked for their purity by TLC, HPLC, elemental analysis, and
mass spectrometry. 2-Chlorotritylchloride resin (1.3 mmol/g) was purchased from
Calbiochem–Novabiochem AG (Switzerland).
General Methods
TLC of the protected amino acids and peptides prepared was performed on
precoated Silufol254 plates (Kavalier, Czech Republic) in the following systems: 2-
butanol–98% formic acid–water (75 : 13.5 : 11.5) (S1); 2-butanol–25% aqueous am-
monia–water (85 : 7.5 : 7.5) (S2); 1-butanol–acetic acid–water (4 : 1 : 1) (S3); and 1-
butanol–pyridine–acetic acid–water (15 : 10 : 3 : 6) (S4). The compounds were visual-
ized directly under an ultraviolet light or by ninhydrin and chlorine–TDM detection
(6). Melting points were measured on a Kofler block and were not corrected.
Solvents were evaporated in vacuo on a rotary evaporator (bath temperature 30ЊC);
DMF was evaporated at 30ЊC and 150 Pa. Analytical electrophoresis was carried
out in a moist chamber on Whatman 3MM paper (20 V/cm) in 6% acetic acid (pH
2.4) and in a pyridine–acetate buffer (pH 5.7) for 60 min. Optical rotations were
obtained on a Perkin–Elmer 141 MCA polarimeter at 20ЊC. The samples for amino
acid analysis were hydrolyzed by 6 M HCl at 110ЊC for 20 h with 3% phenol. The
amino acid analyses were performed on a Durum D-500 amino acid analyzer (Du-
rum Instruments, USA). The molecular weights of the peptides were determined
using mass spectrometry with FAB technique (VG Analytical, England). For HPLC,
a Spectra Physics instrument with an SP 8800 pump, an SP 8450 UV detector, and
an SP 4290 integrator was used. Peptides 1a–1g were purified by preparative HPLC
on a 35 ϫ 2.5-cm RP-18 column, 10-Ȑm (Vydac, The Separations Group, Hesperia,
USA). Analytical HPLC was carried out on a 250 ϫ 4-mm RP-18 column, 5-Ȑm
(LiChrospher WP-300, Merck Darmstadt, Germany). The samples for the elemental
analysis performed at the analytical department of the above institute were dried
over P2O5 at room temperature at 150 Pa.
Syntheses
tert-Butyloxycarbonyl-prolyl-alanine methyl ester (2a). A solution of Boc-Pro-OH
(13.8 g; 70 mmol), HOBt (10.3 g; 77 mmol), and DCC (14.4 g; 70 mmol) in DMF
(40 ml) was stirred at Ϫ10ЊC for 30 min and then filtered to a cooled solution of
HCl.H-Ala-OMe (10 g, 72 mmol) in DMF (10 ml) and pH 7.5 adjusted by DIEA.
The reaction mixture was stirred for 2 h at 0ЊC followed by stirring overnight at
room temperature. The mixture was then cooled in refrigerator and the precipitated
dicyclohexylurea separated by filtration and DMF evaporated. The residue was
dissolved in ethyl acetate (EtOAc) and the solution washed three times with 1 M
NaHCO3 , H2O, 20% citric acid, and a saturated solution of Na2SO4 . After drying

´ ˇ
HLAVACEK ET AL.
134
by anhydrous Na2SO4 the EtOAc solution was evaporated and the resulting product
crystallized from EtOAc–light petroleum to give 13.4 g of the protected dipeptide 2a
with m.p. 77–80ЊC. [Ͱ]D Ϫ57.7Њ (c 0.5, DMF), Ϫ93.3Њ (c 0.4, MeOH). For C14H24N2O5
(300.4) calculated: 55.99% C, 8.05% H, 9.33% N; found: 55.78% C, 8.01% H, 9.21%
N. Mass spectrum (FAB), m/z 301.2 (Mϩ ϩ1). Amino acid composition: Ala (1),
Pro (1.02).
N-Benzyloxycarbonyl-O-tert-butylaspartyl-prolyl-alanine methyl ester (2b). The
Boc-Pro-Ala-OMe (2a, 6.5 mmol, 1.95 g) was treated with TFA (10 ml) for 30 min
2.4
2.4
5.7
and the resulting TFA и H-Pro-Ala-OMe (E
1.44; E
0.95; E
0.99) was
Gly
His
His
acylated by Z-Asp(OtBu)-OSu (7 mmol, 2.8 g) in DMF (15 ml) in the presence of
HOBt (6.5 mmol, 0.87 g) at room temperature and pH 7 was maintained by DIEA.
The reaction mixture was worked up after 2 days similarly to preparation of 2a to
give 3.9 g of the protected tripeptide 2b. The pure product with m.p. 103–105ЊC
was obtained on crystallization from EtOAc–light petroleum. [Ͱ]D Ϫ67.2Њ (c 0.3,
DMF). For C25H35N3O8 (505.6) calculated: 59.39% C, 6.98% H, 8.31% N; found:
59.32% C, 7.01% H, 8.45% N. Mass spectrum (FAB), m/z: 506.2 (Mϩ ϩ1). Amino
acid composition: Asp (1.02), Ala (1.0), Pro (0.95).
N-tert-Butyloxycarbonyltyrosyl-O-tert-butylaspartyl-prolyl-alanine methyl ester
(2c). The Z-Asp(OtBu)-Pro-Ala-OMe (2b, 2.02 g, 4 mmol) in MeOH (100 ml) was
hydrogenated in the presence of Pd black for 3 h, and the resulting H-Asp(OtBu)-
2.4
2.4
5.7
Pro-Ala-OMe (E
0.99; E
0.67; E
0.76) was acylated by Boc-Tyr-OSu
Gly
His
His
(4 mmol, 1.5 g) in DMF (15 ml) in the presence of HOBt (4 mmol, 0.54 g) for 2
days. The reaction mixture was worked up similarly to the synthesis of 2a with a
yield of the amorphous 2c (2.4 g). TLC: 0.73(S1), 0.77(S2). For C31H46N4O10 (634.7)
calculated: 58.66% C, 7.30% H, 8.83% N; found: 58.44% C, 7.41% H, 8.97% N. Mass
spectrum (FAB), m/z 635.3 (Mϩ ϩ1). Amino acid composition: Tyr (0.97), Asp
(1.02), Ala (1.0), Pro (1.01).
N-tert-Butyloxycarbonyltyrosyl-O-tert-butylaspartyl-prolyl-alanine (2d). The solu-
tion of the Boc-Tyr-Asp(OtBu)-Pro-Ala-Pro-OMe (2c, 1.27 g; 2 mmol) in acetone
(30 ml)–water (3 ml) was stirred with 2 M NaOH (1 ml; 2 mmol) at room temperature
for 3 h. Water was added (10 ml), acetone evaporated, and the alkaline solution
washed with EtOAc and acidified by 20% citric acid to pH 3.5. The oily product
was taken to EtOAc and the EtOAc solution washed by water, dried by anhydrous
Na2SO4 , and after filtration evaporated to dryness. The residue was solidified from
ether–light petroleum to give the protected tetrapeptide acid 2d (0.98 g) with TLC:
0.57 (S2), 0.62 (S4). [Ͱ]D Ϫ112Њ (c 0.4; DMF). For C30H44N4O10 (620.7) calculated:
58.05% C, 7.15% H, 9.03% N; found: 57.81% C, 7.04% H, 9.29% N. Mass spectrum
(FAB), m/z 621.3 (Mϩ ϩ1). Amino acid composition corresponded to that of the
methyl ester 2c.
Tyrosyl-aspartyl-prolyl-alanine (1a). The protected tetrapeptide 2d (0.19 g; 0.3
mmol) was dissolved in a mixture of TFA (5 ml)–anisole (0.5 ml) at room tempera-
ture and after 1 h the solution was evaporated and the residue dissolved in 10%
AcOH (200 ml), washed three times with ethyl acetate, and freeze dried. The
peptide was purified by preparative HPLC on a 30 ϫ 2.5-cm column filled with
Vydac stationary phase with a gradient of 5–20% ACN in 0.05% TFA in 30 min

ACCELERATED ACTIVITY OOSTATIC HORMONE ANALOGS
135
TABLE 1
Analytical Data on Peptides 1a–1c
AAAc
E2.4,d
Egly Ehis
Compounda
Formulab
E5.7
EPic
HPLCe
MW/(Mϩ ϩ 1)
Tyr
Asp Ala Pro
HTyrAspProAlaOH
8.59
(1a)
C21H28N4O8
464.8/465.3
C26H35N5O9
561.2/562.2
C31H42N6O10
658.7/659.2
1.03 1.01
1.02 1.02
1.04 1.03
1.0
1.0
1.0
0.98
0.75 0.51 0.29
0.72 0.48 0.27
0.68 0.44 0.24
HTyrAspProAlaProOH
11.80
(1b)
2.1
HTyrAspProAlaPro2OH
14.17
(1c)
2.9
a Analytical data of peptides 1d–1g, see (4).
b Determined with FAB technique (VG Analytical, England).
c Amino acid analyses were performed on a Durum D-500 AA analyzer (Durum Instruments, Corp.,
Palo Alto, CA).
d Analytical electrophoresis was carried out in a moist chamber on Whatman 3MM paper (20 V/cm)
in 6% acetic acid (pH 2.4) and in a pyridine–acetate buffer (pH 5.7) for 60 min.
e Retention time in minutes, 25 ϫ 0.4-cm RP-18 column, 5-Ȑm (LiChrospher WP-300, Merck Darm-
stadt, Germany), flow rate 60 ml/h, detection at 222 nm, gradient 5–50% of acetonitrile in 0.05% aqueous
TFA, 25 min; Spectra Physics 8800 HPLC pump with SP 8450 UV/VIS detector and SP 4290 integrator.
and characterized by mass spectrometry, amino acid analysis, electrophoresis, and
analytical HPLC (Table 1).
Solid-Phase Synthesis of Peptide 1b
The solution of Fmoc-Pro-OH (0.92 g; 2.73 mmol) in dry DCM (30 ml) containing
2 ml of dry DMF (just enough to make the amino acid dissolve in DCM) was added
to the 2-chlorotritylchloride resin (3 g) together with about one-third of the total
amount (1.2 ml, 7 mmol) of DIEA. After 5 min of stirring the remainder of the
DIEA in DCM (1 : 1) was added. Vigorous stirring was prolonged for an additional
60 min. At the end of this time an excess of HPLC-grade MeOH (3 ml) was added
and stirring continued for 10 min to end cap the remaining trityl groups on the
resin. The resin was then washed three times with 30-ml portions of DCM, DMF,
methanol, and diethylether and dried over KOH in a dessicator (3.6 g).
The Fmoc-Pro-2-chlorotrityl resin (0.6 g) was swollen in DMF/DCM (1 : 1) and
deprotected by 5% piperidine in DMF for 10 min (30 ml) followed by 20% piperidine
in DMF (30 ml) for 15 min. The free amino acid resin was then gradually acylated
with three equivalents of Fmoc-Ala-OH (0.29 g), Fmoc-Pro-OH (0.31 g), Fmoc-
Asp(tBu)-OH (0.38 g), and Fmoc-Tyr(tBu)-OH (0.43 g) activated by HOBt/DCC
mixture (0.19 g, 1.4 mmol/0.21 g/1.02 mmol) in DMF (30 ml). At each step cleavage
of the Fmoc group has been done by 20% piperidine in DMF (2ϫ 30 ml, 30 and

´ ˇ
HLAVACEK ET AL.
136
45 min). Finally, the TFA/anisole mixture (10 ml; 9 : 1) was used to carry out the
simultaneous cleavage of the side-chain tBu protecting groups and peptide 1b from
the resin.
The Fmoc-Pro-2-chlorotrityl resin was further treated similarly to the preparation
of 1b using corresponding amino acids with regard to a number of Pro residues in
the C-terminus of the resulting peptides 1c–1g.
Peptides 1b–1g were purified and characterized under the same conditions used
for peptide 1a. The analytical data of the new peptides 1a–1c are shown in Table
1. Those of the newly synthesized peptides 1d–1g were identical to data of the
peptides synthesized early by a solution method as described in our previous pa-
per (4).
Iodination with 125I
A solution of corresponding peptide (5 mg) in 0.05 M sodium phosphate buffer,
pH 7.5 (25 ml), was placed in an IODO-GEN tube (7) washed earlier with this
phosphate buffer. To this solution the 10 ml of Na125I (corresponding to 37 MBq)
was added and after 60 min the reaction was stopped by transporting the reaction
mixture out of the IODO-GEN tube to a test tube and adding the 35 ml of 0.05
M KI. Separation of the iodinated peptide from unreacted Na125I was performed
on a column of Sephadex G-25 (55 ϫ 1.4 cm) which was equilibrated with 0.02 M
sodium phosphate buffer.
Bioassays and Radiotracer Distribution Studies
The peptides under study were separately injected in Ringer solution (1 Ȑg/ml,
3 Ȑl per female) into the upper part of the thorax of freshly emerged adult females
of the flesh fly Sarcophaga bullata Parker (Diptera). The flies were dissected at
regular time intervals (on days 1, 2, 7, 8, and 15 after application) and their ovaries
were examined for changes in egg development during the first gonotrophic cycle.
Egg hatchability was determined on deposited eggs in the uterus. The ovaries
undergoing the second gonotrophic cycle were examined as well. Histological prepa-
rations made of suspect ovaries of both gonotrophic cycles were prepared by stan-
dard procedure. Differences in the degree of inhibition of correct egg development
were evaluated with regard to the regular time intervals after peptide application
and the peptide length. A general relationship between a decreasing number of
the C-terminal Pro residues in peptides 1a–1g and the beginning of the oostatic
effect was evaluated as an acceleration phenomenon.
For radioactivity microdistribution within an insect body, a dissected organ or
an appropriate body part was dissolved in 1 ml of a scintillation cocktail (Soluene
350, Packard) using shaking at 40ЊC for 48 h. Liquid scintillation spectrometry
(Beckman, LS 6500) was then used for radioactivity measurement.
RESULTS AND DISCUSSION
For the synthesis of peptides 1b–1g the solid phase approach was chosen. After
the first Fmoc-Pro residue was bound to the resin and the Fmoc protecting group
was split off by 20% piperidine in DMF, the polymer material was used for synthesis

ACCELERATED ACTIVITY OOSTATIC HORMONE ANALOGS
137
SCHEME 1. Synthesis of tetrapeptide 1a.
of the corresponding peptides with a gradually growing chain. The solid-phase
synthesis was carried out with the 2-chlorotrityl linker which is particularly suited
to the synthesis of the Pro peptides. The extreme steric hindrance of the trityl group
means that formation of dioxopiperazine at the dipeptide step, with consequent
loss of peptide from the resin, is totally suppressed.
Tetrapeptide 1a without the C-terminal Pro residue was prepared in solution
similarly to the first series of our oostatic peptides described in our previous paper
(4) (Scheme 1).
Morphology and Histology
Using tetrapeptide 1a and pentapeptide 1b for application, morphological and
histological observations revealed changes in ovarian development. Morphologi-
cally affected egg chambers have irregular appearance; their shape is often distorted.

´ ˇ
HLAVACEK ET AL.
138
Histological evaluation revealed the following events: (1) a division of the nucleolus
of the follicular cell which is followed by nuclear division and cell division; and (2)
the rapid cell division and migration of such cells into yolk as well as into a region
of nutritive cells, followed by a complete desintegration of the yolk and nutritive
cells. In this way, the whole inner space of the egg chamber is filled up by those
proliferating cells. Finally, resorption of such an affected egg chamber occurred.
The occurrence of those changes varies according to the length (a number of the
Pro residues) of the C-terminus for the whole series of the peptides investigated
(1a–1g). A shortening of the peptide molecules from the C-terminus was followed
by reaching the given biological effect in the shorter time period. The changes
appear mostly in the development of the second batch of eggs; the changes in the
first gonotrophic cycle are manifested by decreased hatchability of eggs in the
uterus. For example, comparing decapeptide 1g which allows up to 80% of egg
hatchability, the pentapeptide 1b application decreases egg hatchability to 40% and
that of tetrapeptide 1a up to 20%. As a result the tetra- and pentapeptides 1a and
1b were found to effect more ovarioles in comparison to the larger peptides 1c–1f
and even more to the native decapeptide 1g due to reaching the oostatic effect at
the beginning of the bioassay. On the other hand, larger peptides 1c–1g including
the native one needed more time to evoke a similar biological effect.
Comparing the effects of tetrapeptide 1a and pentapeptide 1b with those of the
larger peptides 1d–1g, the final effect after day 15 is the same—resorption, but the
initial changes—proliferation and cell migration detected within the first period of
the bioassay (the first gonotrophic cycle)—are most pronounced using tetrapeptide
1a and pentapeptide 1b. It follows from a comparison to the previously assayed
peptide series including decapeptide 1g (4) that the shortest peptides reveal signifi-
cantly accelerated activity (8).
Radioactivity Microdistribution
The radiolabeled forms of tetrapeptide 1a and pentapeptide 1b with the most
pronounced oostatic effect were used to describe the transportation of the applied
radioactivity through the insect body and its transfer from the treated female body
to the next (F1) generation. Such investigations could considerably contribute to
analyses of the mode of action of the peptides under investigation within the treated
body and to the description of the transfer to the next generation including that
affecting of this generation (8).
It follows from the results given in Table 2 that the excretion of tetrapeptide 1a
shortly after its application is more intensive than that of pentapeptide 1b under
the same experimental conditions. Therefore, a lower radioactivity remains available
for distribution within the insect body during days 1 and 2 after application of
tetrapeptide. Nevertheless, as it was described, proliferation and cell migration
representing the initial effects are more pronounced using tetrapeptide. All obtained
findings so indicate an accelerated activity of tetrapeptide in comparison to penta-
peptide. Twenty-four hours after separated application of both radiolabeled pep-
tides, radioactivity was already detected in all other analyzed body parts, i.e., head,
fat body, and ovaries.

ACCELERATED ACTIVITY OOSTATIC HORMONE ANALOGS
TABLE 2
139
Relative Mean Radioactivities (in Percentage of the Applied Radioactivity) after Application of the
Radiolabeled Tetrapeptide (T) or Pentapeptide (P), Respectively
Days after application
Peptide
1
2
7
15
T
P
T
P
T
P
T
P
Gut
21.3
17.3
10.1
4.7
8.9
4.6
0.7
4.5
7.4
1.6
5.5
6.4
Uterus with eggs
Uterus with larvae
Note. N ϭ 10. SD of each radioactivity measurement ϽϮ1%.
The main result of the paper on freshly emerged Sarcophaga bullata female flies
is to demonstrate that the development of the egg chamber and yolk deposition
were affected by the injection of both the oostatic hormone and peptide sequences
with a lower number of proline residues, in that way, shortening of the carboxy
terminus enhances the observed effects by affecting the first gonotrophic cycle as
well and thus shortens the time interval after injection at which time the morphologi-
cal changes appear.
From this it could be suggested that the oostatic hormone with the six C-terminal
proline residues can play, under physiological conditions, a role of hormonogene
or prohormone. A space arrangement of the proline residues could exhibit an
important role in active sequence protection against premature enzyme degradation
during peptide transport to a corresponding receptor and even more in binding of
the peptide to this receptor. Once a conformation of the decapeptide is changed
within a complex hormone receptor, an active amino-terminal tetrapeptide part
can be released for the next biological function including a transduction of the
biological effect. Our shortened peptides, especially tetrapeptide 1a without C-
terminal proline residues, could participate in biological activity transduction di-
rectly, thus accelerating oostasis and also causing total damage and resorption of
the egg.
As to the radiolabeling study performed and described in our paper, the main
goals of application of the radiolabeled peptides were:
(1) To prove different changes in radioactivity distribution within an insect
body in different time intervals after injection. In this way it has been relatively
simply demonstrated that the kinetics of the different labeled peptide molecules
under study in the insect body are different. The determination of the radioactivity
distribution within the fly body as an introductory act was important for seeing
what part of the body is worth detailed structure analysis of the isolated peptide
material to be performed during further research.
(2) To prove that the radioactivity is transported to the next F1 generation of
the treated flies. The radioactivity transfer to male or female body parts of the F1
generation, respectively, was proven on one side by measurement of the radioactivity
of the uterus with eggs and larvae of the treated fly (Table 2) and, on the other,

´ ˇ
HLAVACEK ET AL.
140
by the radioactivity measurement of the males and females of the F1 generation,
respectively. The difference between male and female body parts was not significant
for both peptides applied. The mean of 8-day-old flies (n ϭ 10) was found to
be 0.03% of the radioactivity applied to the ‘‘mother’’ body. This value roughly
corresponds to the value of the uterus given in Table 2.
(3) To find out whether peptides with an iodinated tyrosine residue (either
radio- or non-radiolabeled) trigger similar oostatic effects as the peptides containing
the tyrosine residue only. Our preliminary results suggest that iodination of the
tyrosine probably does not interfere with recognition and binding of corresponding
peptides to a receptor.
We consider the analysis of the radiolabeled degradation products as a next step
which represents a different experimental approach, especially due to very low
amounts of the analyzed fractions after radiochromatography and to the necessity
of their isolation with sufficient high purity for identification. Our previous experi-
ments and analytical measurements indicate some modification of the peptide mole-
cule structures during peptide application and their action in biological material.
ACKNOWLEDGMENT
This study was supported by grant of the Grant Agency of the Czech Republic (505/95/1030).
REFERENCES
1. DeMilo, A. B., Kelly, T. J., Masler, E. P., and Bell, R. A. (1991) in Insect Neuropeptides: Chemistry,
Biology, and Action (Menn, J. J., Kelly, T. J., and Masler, E. P., Eds.), pp. 143–151, Am. Chem.
Soc., Washington, DC.
2. Borovsky, D., Carlson, D. A., Griffin, P. R., Shabanowitz, J., and Hunt, D. F. (1990) FASEB J.
4, 3015–3020.
3. Nomenclature and Symbolism for Amino Acids and Peptides. Recommendations 1983 (1984) Eur.
J. Biochem. 138, 9–37.
4. Hlavacek, J., Bennettova, B., Barth, T., and Tykva, R. (1997) J. Pept. Res. 50, 153–158.
´ ˇ
´
5. Wunsch, E. (1974) in Synthese von Peptiden, Teil I. Methoden der Organische Chemie (Houben-
¨
Weyl), pp. 46–404, Georg Thieme Verlag, Stuttgart.
6. Von Arx, E., Faupel, M., and Brugger, M. (1976) J. Chromatogr. 120, 224–228.
7. Fraker, P. J., and Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80, 849–857.
8. Bennettova, B., Hlavacek, J., and Tykva, R . (1997) The Abstract Book of the International Conference
´ ˇ
´
on Invertebrate Neurochemistry and Neurophysiology, 1997, Eilat, Israel.