have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 ...
Biochimie (1991) 73, 1233-1239 © Soci6t6 frangaise de biochimie et biologie mol6culaire / Elsevier, Paris
1233
Structure and biological activity of crustacean gastrointestinal peptides identified with antibodies to gastrin/cholecystokinin P Favrel , G Kegel2, D Sedlmeier2, R Keller2, A Van Wormhoudtm tLaboratoire de Biologic Marine du ColiOge de France, 29900 Concarneau, France; 2hlstitutfiir Zoophysiologie, Univer~itiit Bonn, Endenicher Allee ! !-13, 5300 Bonn, Germany (Received I I January 1991; accepted 23 May 199 l)
Summary - - Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum
have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans. gastrin/CCK / stomach / aminoacid composition / crustacea
Introduction
In recent years, numerous immunochemical and biochemical data have provided substantial evidence indicating that vertebrate biologically active peptides occur in various invertebrate species, from the most primitive ones, like protozoans [1, 2], to protochordates [3]. Little is k n o w n about the structure of these molecules except for that of the well-known examples of the insulin-like substances [4-6] and the vasopressin/oxytocin analogues [7]. The presence of substances structurally related to the vertebrate gastrin/cholecystokinin (G/CCK) family of regulatory peptides has also been well demonstrated in different phyla [8]. Only insect peptides with homologies to G / C C K have been sequenced so far. They include leukosulfakinins I and II, two myotropic neuropeptides extracted from the cockroach [9, 10], drosulfakinins whose sequence was deduced from that of the corresponding c D N A [1 1], the perisulfakins from Periplaneta [ 12] and the sulfakinins from Leucophaea [13]. However, these peptides are fundamentally different from the genuine CCK/gastrins and should be regarded as part o f a different family. W e previously described the characterisation of gastrin/CCK-like peptides in the prawn Palaemon
serratus using an antiserum specific to the C-terminal sequence c o m m o n to gastrin and C C K [ 14]. We report here the isolation and the aminoacid composition of some G/CCK-like peptides extracted from the stomach of another crustacean: the Norway lobster Nephrops norvegicus. Since these peptides appear to control digestion processes in crustaceans [15, 16], their involvement in the release of digestive enzymes has been investigated. Materials and methods
Animals Norway lobsters, Nephrops norvegicus were bought in a local fishery (Concarneau, France). They were kept one night before use in an aerated sea water tank. Crayfish, Orconectes limosus was supplied by a commercial fisherman from the Havel river near Berlin-Spandau and kept in compartmented aquaria with constant water flow (12-15°C).
Radioimmunoassay The development of the radioimmunoassay (RIA) has previously been described [14]. The antibody (kind gift of Dr G Tramu, INSERM, U 156, Lille, France) was specific for the C-terminal pentapeptide common to the vertebrate gastrins and cholecystokinins. In brief, the fractions were subjected to
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P Favrel et al
G/CCK RIA using 125I-labelled gastrin 17-I purchased from Oris (Gif-sur-Yvette, France) as tracer (specific activity: 1800 Ci/mmol) and synthetic human gastrin 17-I (G 17-1) as standard. Samples containing the antiserum at a final dilution of 1/I 800 000, the labelled hormone and the suitably diluted biological extracts were incubated at 4°C for 48 h in a 0.02 M veronal buffer, pH 8.4, containing 1% BSA. The free-labelled peptide was separated from the antibody-125I-gastrin complex by charcoal adsorption (SXX extra).
Aminoacid analysis The purified fractions (400 pmol, as deduced according to their absorbance rate at 226 nm) were hydrolysed in vacuo in sealed glass tubes with 50 BI of 5.7 M HC! (Pierce) containing 0.4% ~-mercaptoethanol during 24 h at 110°C. Analyses were performed with a Biotronic LC 5000 analyser using BTC 2710 resin and a citrate/borate buffer as previously described [ 17].
End-group determination The N-terminal residue was identified according to Chang [ 18].
Extraction Hydrolysis with the pyroglutamate aminopeptidase 200 g of stomachs (equivalent to 20 kg of animals) were excised, rinsed with sea-water and immediately frozen in liquid nitrogen until extraction. The extraction was carried out in 10 volumes of deionised, boiling water for 15 min and sonicated. The extract was centrifuged tbr 15 min at 18 000 g and the pellet was re-extracted in 0.5 i of deionised water. After a final centrifugation, the supernatants were freeze-dried.
1 lag of peptide E was dissolved in 80 BI potassium phosphate buffer 0. I M, pH 8 containing 5 mM DTT, 5 mM EDTA and 5% glycerol containing 40 lag of pyroglutamate aminopeptidase (Boehringer, Mannheim, Germany). After 1 h incubation at 30°C, the reaction was stopped with 80 lal of 4 M acetic acid, centrifuged 5 min at 10 000 g and directly applied to the Vydac C~s column.
Purifi¢'ation
Amino acid sequence determination
Molecular sieving The freeze-dried extract was dissolved in 30 ml of 10 mM ammonium acetate buffer (pH 8.5) and cleared by ultracentrifugation during 10 min at 30 000 g (Beckman L 8.70). It was subsequently divided into three batches (3 x 10 ml) that were subjected to a molecular sieving on a Sephadex G50 fine (Pharmacia, Sweden) column (2.5 x 100 cm) previously equilibrated with 10 mM ammonium acetate. The immunoreactive fractions were pooled and lyophilised.
Sep-pak separation The lyophilised fractions were redissolved in water containing 0.1% TFA and applied to Sep-pak cartridges (C~s, Waters Associates). The adsorbed material was eluted with 0.1% TFA in 70% n-propanol.
Reverse phase high performance liquid chromatography (RPHPLC) HPLC was performed on a LKB (Sweden) model 2150 liquid chromatograph equipped with a Rheodyne 7125 sample injector and a LKB 2158 Uvicord wavelength detector. ODS column. After evaporation, the material was dissolved in 17% n-propanol 0.1% TFA (w/v) = A and subjected to a RPtiPLC C~s column (Altex 5 lam, 24 × 0.46 cm). The elution was carried out using 100% A during 40 min, then with a linear gradient up to 35% B in 100 min at a flow rate of 0.5 ml/min (solvent B: 70% propanol, 0.1% TFA (w/v)). Phenyl microbondapak column. The fractions containing the highest amount of immunoreactive material were dissolved in 0.11% TFA (solvent A) and rechromatographed on an alkyiphenyl microbondapak column (Waters Associates, 5 Bm, 0.39 x 30 cm) with a linear gradient from 30 to 80% B, in 60 rain (solvent B: 0. ! % TFA, 60% acetonitrile) at a flow rate of 0.9 ml/min. Nucleosi1300 A and Vydac peptide Cts cohmm. The G/CCKlike immunoreactive fractions identified in the previous mep were loaded onto another reverse phase C~s HPLC column (Nucleosil 3 Bm, 0.46 x 25 cm, CIL, Sainte-Foix, France) and finally onto a C~s protein/peptide HPLC column (Vydac 3 l.tm, 0.46 x 25 cm, Sep group, Hesperia, USA). Elution was achieved using the same solutions as for the alkyl-phenyl column with a modified gradient.
About 1 nmol of pure peptide DI was subjected to sequence analysis by a standard method using an Applied Biosystem gas phase sequenator (CNRS, Vemaison, France).
Biological assay Secretagogue effects Orconectes limosus midgut glands were dissected out and washed in Harrefeld saline as previously described [191. A piece of gland thoroughly rinsed was placed into a tube containing 2 ml of saline solution. Substances to be tested were added in a volume of 10 lal after a preincubation period of 10 min. Incubation was performed at 25°C for 2 h. Protease activity in the incubation medium was measured according to Charney and Tomarelli 1201 after the preincubation period and after 2 h of further incubation. Also the remaining protease in the midgut gland sample was determined after the 2-h incubation period. Total protease was calculated after summation of these 3 values. Results are expressed as percentage of protease released from total protease.
Results Purification The 200 g s t o m a c h extract o f Nephrops y i e l d e d an a m o u n t o f G / C C K - I i k e m a t e r i a l w h i c h a c c o r d i n g to the R I A results was e q u i v a l e n t to 4 6 n g o f G I7-I. T h e f r a c t i o n a t i o n o f this s a m p l e o n a S e p h a d e x G 5 0 (fine) c o l u m n s h o w e d the p r e s e n c e o f four m a i n G / C C K i m m u n o r e a c t i v e c o m p o n e n t s (fig 1): a h i g h m o l e c u l a r w e i g h t f o r m (A) e l u t i n g close to the v o i d v o l u m e a n d three s m a l l e r f o r m s (C, D, E) with apparent m o l e c u l a r w e i g h t s r a n g i n g f r o m 2 0 0 0 to 1000 Da. T h e m a t e r i a l in the fractions C, D, a n d E was further purified b y a series o f H P L C steps. D u r i n g the initial s e p a r a t i o n on the Altex-C~8 c o l u m n , the three fractions E, D, a n d C eluted w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f n-proi::,,~l. Some i m m u n o r e a c t i v i t y wcs also d e t e c t e d for t]',t: fractions
Crustacean gastrointestinal peptides A 226
1235 %B
nm
9
o
~ . s -
ag ? ~~
.z,,,= 1 0
.5-
A
o .=
4 3 2
Volume ( ml )
Fig 1. Gel filtration of stomach extract from Nephrops norvegicus and identification of gastrin/CCK-like immunoreactive components. Molecular sieving of 10 ml sample performed on a Sephadex G50F column (100 × 2.5 cm), eluted at 4°C with 10 mM ammonium acetate pH 8.6 at a flow rate of 30 ml/h. Absorbance was monitored at 280 nm and immunoreactivity was measured from aliquots from each tube.
C and D in the void volume, but disappeared after rechromatography and afterwards eluted at a normal position (data not shown). Next, each fraction was reinjected on an alkyl-phenyl-microbondapak column. Two peptides were obtained from the fractions C and D, respectively, while only one peptide emerged from the fraction E (fig 2). Purification was continued on the main fractions, ie fractions C1, C2, D1 and E. After two other steps of purification on a Nucleosil-Cma column and a VydacCm8 column, highly purified peptides were recovered: single homogenous peaks appeared to elute at different acetonitrile concentrations (fig 3).
Amino acid analysis Amino acid analysis of the peptides obtained after the final step is shown in table I. Because the antibody used requires a phenylalanine:amide residue to fully cross-react, we calculated the amino acid ratios by assuming the presence of at least one mole of this residue per mole of peptide. The calculation was based on two separate batches of material which gave very similar amino acid compositions. Six amino acids resndues (Asx,i Ser, Glx, 2 Gly, Phe) were common to all peptide'J (table I). Peptide •
.
.
•
/
i
I
.5
50
02
01
•
I
I
I
••o
0
,b
l
• •0•
••••
• o•
••°••
•
,b Volume (ml)
Fig 2. Reverse phase high performance liquid chromatography on an alkylphenyl microbondapak column (Waters, 5 Hm, 0.39 x 30 cm). Rechromatography, on an alkylphenyl microbondapak column, of the fractions C, D, E obtained after HPLC on a Cn~ (Aitex) column (see Material and methods). A linear gradient of acetonitrile from 30 to 80% in 60 min was applied• Cl, C2 and Dl, D2 correspond to fractions C and D respectively, separated during the preceding steps• D I appeared pure while some contaminants were still present for E, C2 and C1. We noticed a very large discrepancy between the amount of material estimated by RIA and the amount deduced from amino acid
1236
P Favrel et al
%B
A226nm
A226nm
.
° ~s
C2
Cl
m• •
i0
.05
•• ,
•
•••
°•••••O
L.---"
.02
.--"'"
J !
'
,,!
m v
,i
!
|
!
DI .03" 50 .°,P
,02"
I..J..--"
.02, D• • •••
,01LJ.
io
:/o
"""
~--~~---3~0
-"i
20
40
"
k
,
do
3b
t ime (mn)
Fig 3. Final step of purification of Nephrops G/CCK peptides on a Vydac HPLC column (3 lam, 0.46 x 25 cm). Table !. Amino acid composition of tour Nephrops gastrin/CCK-like peptides. The values are expressed in mol %. Numbers in parentheses were calculated on the basis of I mole phenylalanine per mole of peptide and rounded up to the nearest integer, nd: not determined,
Asx Thr Ser GIx Gly Ala Val lie Leu Met Tyr Phe His Lys Arg Pro Trp* Cys A**
C!
C2
DI
E
8.0 (1) 2,3 6,1 (!) 9.5 (2) 12.9 (2) 5.2 (1) 4,5 (1) 2.5 6.9 (!) 0,9 1.3 6.1 (1) 3.4 2.0 0,0 27.0 (4) 0.2 0.0
8,7 (1) 2,9 5.0(1) 8.3 (1) 13,5 (2) 5.4 (I) 5.7 (1) 4,2 8,6 (!) 0.9 2.2 7,7 (!) 1.3 2.2 1.8 22.0 (3) 1.1 0.0
I 1.0 (I) 0.2 8.0(!) 23.0 (2) 23.4 (2) 0.0 0.0 0, ! 5 11.2 (1) 0.9 0.0 12.3 (I) 0.0 0.0 0.0 0.0 8.9 (1) nd
8.0 (!) 1.4 12.1 (1) 8.0 (1) 16.9 (2) 8.9 (1) 3.0 2. I 2.0 0.8 8.6 (!) 5.4 (1) 3,4 2.0 1.2 2.8 9.8 (1) nd
*Determined after 6 N HCI hydrolysis in the presence of 0.4% [~-mercaptoethanol; **determined after reduction and alkylation.
Crustacean gastrointestinal peptides analysis (table II). The molecular weights estimated from amino acid composition (9 residues for peptides E to 14 residues for C1) were in agreement with the G50 molecular weight estimations.
Digestion with the pyroglutamate aminopeptidase The determination of the N-terminal sequence was done on peptides C, D and E. Serine was found to be present in peptide D I while peptides C and E (1 l.tg) did not yield any residue for the first three cycles of Edman's degradation. An enzymatic digestion with the pyroglutamate aminopeptidase was tried without success although under the same conditions, the pGlublocked AKH was cleaved.
Amino acid sequence A comparison of the complete sequence of Nephrops peptide D 1, human CCKI 2, insect Drosulfakinins and leucosulfakinins is shown in table III.
1237
Discussion
The G/CCK-Iike peptides identified so far in various species of crustacean exhibit some common features with the vertebrate G/CCK system proposed by Rehfeld [21]. They show homologies as demonstrated by immunological cross-reactivities or by receptor binding data [14, 22], they present a certain degree of heterogeneity, they are present in various tissues, including the stomach, the carapace, the hemolymph and the nervous system [ 14, 22-24]. Moreover different molecular forms predominate in different tissues and species. The G50 and Altex C~8 elution profiles of Nephrops stomach extracts are consistent with our previous results concerning Palaemon stomach extracts [ 14]. In both species at least three small molecular weight fractions were detected. From different immunocytochemical studies, it can be concluded that these components derive from the epithelial cells of the
Protease release The release of protease from the midgut glands was significantly increased after the addition of fraction C I or E to the incubation medium at a concentration estimated to be | 0 nM (fig 4). By contrast, fraction D did not seem to stimulate a significant protease release from the digestive gland. Table II. Percentage of immunocross-reactivity between
the different peptides isolated from Nephrops norvegicus to human G 17-I with the G/CCK antiserum.
Fraction
3O
20
lO
CI
C2
DI
E
0.4
0.7
0.4
1.4
Peptides (ng)2
4200
3500
1520
3800
~raetloas tested
% immuno cross-reactivity
0.01
0.02
0.03
0.04
Fig 4. In vitro stimulation of protease secretion from Orconectes limosus hepatopancreas by Nephrops norvegicus G/CCK-like peptides. Each peptide was tested at a concentration estimated to be 10 nM (n = 6). Mean + SE indicated as a transverse b~,r. Significance of difference with control. ** P < 0.001.
G/CCK-like (ng) I
'Total amount measured by RIA (from fig 3: data not shown). 2Total amount estimated after amino acid hydrolysis.
Cont.
C1
D1
E
Table III. Amino acid sequences of human CCKI2, Nephrops peptide DI, insect Drosulfakinin I and II (DSK I, !I) and insect
Leucosulfakinin I and II (LSK I, II). CCKI2 DI DSK I DSK II LSK I LSK II
Ile-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met- Asp-PheNH2 Ser-Glu-Gly-Gly-Gln- Asp-Phe-Trp-Leu Phe-Asp-Asp-Tyr-Gly-His-Met-Arg- Phe(NH2) Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg- Fhe(NH2) pGlu-Gln-Phe-Glu-Asp-Tyr-Gly-His-Met-Arg- PheNH2 pGlu-Ser-Asp-Asp-Tyr-Gly-His-Met-Arg- PheNH2
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P Favrel et al
stomachs [14, 25]. Immunopositive nervous fibres of the stomatogastric system which show G/CCK-Iike peptides with identical chromatographic properties might also be a possible source [24, 26, 27]. The Nephrops G/CCK immunoreactive peptides exhibit, like their vertebrate relatives, a high degree of molecular diversity. Among the five molecules detected, four were purified. After five steps of purification, only minute amounts of peptide were obtained. The recoveries were poor (estimated around 6%) and can be attributed for fractions C and D to the presence of high amounts of carbohydrate contaminants which co-elute from the G50 column and hence hinder the binding of peptides to the C~s chains during the first RP-HPLC. After the final step of purification, the peptides were considered to be pure (D) by their absorption profile or only slightly contaminated (C1, C2, E) as suggested by their amino acid composition. The average cross-reactivity ratio was estimated to be 0.03% and reflects structural differences between the crustacean and the vertebrate G/CCK antigenic determinants recognised by the antibody. Indeed, among the five amino acid residues (Gly, Trp, Met, Asp, Phe) required for a full immunoreactivity, only three (Gly, Asp, Phe) or four (Trp) are present in the G/CCK-iike molecules. Sequence analysis shows that only Gly, Asp and Phe are present at similar positions in the C~ terminal part of peptide D1 and vertebrate gastrins. This probably explains the very low cross-reactivity observed. Surprisingly, because it lacks histidine, methionine and arginine residues, it also differs considerably from the co-called insect G/CCKs ie from leucosulfakinins I and II [9, 10] and from drosulfakinins [11]. This is understandable to some extent, since our antibody did not react with FMRF-amide up to 10-4 M [14], the sequence of which is also partially present at the C-terminal part of the insect G/CCKs [28]. Such an observation as well as the presence of a C-terminal extension on peptide D1 suggest that this molecule belongs to a novel family of peptides. Interestingly, all the other Nephrops peptides show an homologous amino acid composition: 6 amino acid residues detected in the smallest forms (D 1 and E) are also constitutive residues of the larger ones, C I and C2. The post-prandial variations of these peptides in the hemolymph of penae'fds show the disappearance of a high molecular weight fraction concomitant with the increase in small molecular weight peptides [15]. These observations suggest the existence of a high molecular weight precursor. As is the case for drosulfakinins, several peptides with homologous sequences may well derive from the same precursor [11]. Clearly, further experiments are necessary to support these assumptions. Recombinant DNA technology is likely to bring convincing answers to these questions. As to the biological functions fulfilled by these G/CCK-Iike peptides in crustaceans, there is no clear
answer yet. The vertebrate gastrins and CCKs can, although with high doses, ellicit different biological responses in invertebrates such as the induction of the release of digestive enzymes [29, 30], stimulation of protein synthesis in the midgut gland of the prawn [31 ] or modulation of the gastric mill motor pattern in the lobster [ 16, 24]. Our results strongly suggest that the purified Nephrops G/CCK-Iike peptides C 1 and E stimulate the release of digestive enzymes in vitro. These results agree well with our previous findings of post-prandial related changes of G/CCK in prawns [15]. A greater sensitivity to proteolytic enzymes due to the absence of N-terminal protection for peptide DI may explain its lower biological activity. Alternatively, a kind of specificity among (still unknown) biological functions cannot be ruled out. Recently, the modulation of the output of the stomatogastric system in Palinurus has been reported for one of our peptides (peptide E) [26]. The other peptides tested were not so active in this system and possibly the pr~:~ence of a tyrosine residue in peptide E could be im0ortant for eliciting such a biological activity. The determination of the sequence of the other Nephrops 'G/CCK-Iike' peptides should provide insights into the structural relationship between them and other invertebrate gastrointestinal peptides. Furthermore, synthetic peptides will allow us to define much more deeply their biological function in crustaceans. Acknowledgments
Part of this work was supported by a grant from the 'Association Nationale de la Recherche Technique', Paris, PROCOPE no 88037. References 1 Le Roith D, Shiloach J, Roth J, Leshiak H (1980)
Evolutionary origins of vertebrate hormones: substances similar to mammalian insulins are native to unicellular eucaryotes. Proc Natl Acad Sci USA 77, 6184--6188 2 BerelowitzMD, Le Roith D, Von Schenk H, Newcard C, Szaba M, Frohman LA, Shiloach J, Roth J (1982)
Somatostatin-like immunoreactivity and biological activity is present in Tetrahymena pyriformis, a ciliated protozoan. Endocrinology 110, 1939-1944 3 ThorndykeM, Dockray GJ (1986) Identificationand loealisation of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis. Regul Pept 16, 269-279 4
Nagasawa H, Kataoka H, Isozai A, Tomura S, Suzuki A,
Ishizaki H, Mizoguchi AA, Fugiwara Y, Suzuki S (1984) Amino-terminal amino acid sequence of the silkworm prothoracotropic hormone (PTTH): homology with insulin. Science 226, 1344-1345 5 SmitAB, Vreudenhill E, Ebberink RHM, Geraerts WMP, Klootwijk J, Joose J (1988) Growth-controllingmolluscan neurons produce the precursor of an insulin-related peptide. Nature 33, 535-538
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7
8 9
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11
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15
16 17
Robi¢zki A, Schroder HC, Ugarkovic D, Pfeifer K, Uhlenbruck G, Muller WEG (1989) Demonstration of an endocrine signalling circuit for insulin in the sponge Geodia cydonium. EMBO J 8, 2905-2909 Proux JP, Miller CA, Li .IP, Carney RL, Girardie A, Delaage M, Schooley DA (1987) Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria. Biochem Biophys Res Comm 149, 180-186 Larson BA, Vigna SR (1983) Species and tissue distribution of Gastrin/Cholecystokinin-like substances in some invertebrates. Gen Comp Endocrino150, 469-475 Nachman R.I, Holman GM, Cook B.I, Haddon WF, Ling N (1986) Leucosulfakinin II, a blocked sulphated insect neuropeptide with homology to cholecystokinin and gastrin. Biochem Biophys Res Comm 140, 1,357-364 Nachman RJ, Holman GM, Haddon WF, Ling N (1986) Leucosulfakinin, a sulphated insect neuropeptide with homology to gastrin and cholecystokinin. Science 234, 71-73 Nichols R, Schneuwly SA, Dixon JE (1988) Identification and characterisation of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin. J Biol Chem 263, 12167-12170 Veenstra .IA (1989) Isolation and structure of two Gastrin/CCK-like neuropeptides from the American coakroach homologous to the leukosulfakinins. Neuropeptides 14, 145-149 Schoofs L, Holman F, Hayes TK, De Loof A (1989) Isolation and identification of a sulfakinin-like peptide from the brain of Locusta migratoria with sequence homology to vertebrate gastrin and cholecystokinin. In: Insect hormones (Mc Caffery A, Wilson I, eds) Plenum, NY Favrel P, Van Wormhoudt A, Studler JM, Bellon C (1987) lmmunochemical and biochemical characterisation of gastrin/cholecystokinin-like peptides in Palaemo,: serratus (Crustacea Decapoda): intermolt variations. Gen Comp Endocrino165, 363-372 Van Wormhoudt A, Favrel P, Guillaume J (1989) Gastrin/cholecystokinin-like post-prandial variations: quantitative and qualitative changes in the hemolymph of Penaeids. J Comp Physiol 159, 269-273 Turrigiano G, Selverston AI (1990) A cholecystokininlike hormone activates a feeding-related neural circuit in lobster. Nature 344, 866-868 Keller R (1981) Purification and amino acid composition of the hyperglycemic neurohormone from the sinus gland of Orconectes limosus and comparison with the hormone from Carcinus maenas. J Comp Physiol 14, 445-450
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18 Chang JY (1983) Manual sequencing of polypeptides using DABITC. Methods Enzymol 91,455-466 19 Sedimeier D (1988) The crustacean hyperglycemic hormone (CHH) releases amylase from the crayfish midgut gland. Regul Pept 20, 91-98 20 Charney J, Tomarelli RM (1947) A colonic method for the determination of the proteolytic activity of the duodenal juice. J Biol Chem 171,501-505 21 Larson BA, Vigna SR (1983) Gastrin/cholecystokinin-like immunoreactive peptidcs in the Dungeness crab Cancer magister (Dana). Immunochemical and biological characterisation. Regul Pept 7, 155-170 22 Rehfeld .IF (1981) Four basic characteristics of the gastrin-cholecystokinin system. Am J Physiol 240, G 255-266 23 Van Deynen JEE, Vek F, Van Herp F (1985) An immunocytochemical study of the optic ganglia of the crayfish Astacus leptodactylus with antisera against biological active peptides of vertebrates and invertebrates, 240, 171-183 24 Turrigiano G, Selverston AI (1987) Presence and release of CCK/gastrin-like molecule in the lobster stomatogastric ganglion. Colloq Soc Neurosci Abstr ! 3, 1257 25 Scalise FWW, Larson BA, Vigna SR (1984) Localisation of a peptide identified by antibodies to gastrin/CCK in the gut of Cancer magister. Cell Tissue Res 228, 113-119 26 Turrigiano G, Selverston AI (1989) Cholecystokinin-like peptide is a modulator of a crustacean central pattern generator. J Neurosci 9, 2486-2501 27 Van Wormhoudt A, Dircksen H (1989) Gastrin/CCK-like peptides in the nervous system and the stomach of crustaceans. In: Frontiers in Crustacean Nem'obiology (Wiese K et al, eds) Birkhiiuser Verlag, Basel, 483-484 28 Nachman RT, Holman GM, Haddon WF (1988) Structural aspects of gastrin CCK-like insect leucosulfakinins and FMRF-amide. Peptides 9, 137-143 29 Thorndyke MC, Bevis JPR (1984) Comparative studies on the effect of cholecystokinins, caerulein, bombesin and physalaemin on gastric secretion in ascidian Stylea clava. Gen Comp Endocrino155, 251-259 30 Sedlmeier D, Resch G (1989) Effect of vertebrate peptides on amylolytic and proteolytic activity of crayfish hepatopancreas. XIth Inter Symp Comp Endocrinoi Malaga, 14-20 May 1989 Abstr 315 31 Favrel P (1988) Purification et caract6risation biochimique de peptides immunologiquement apparent6s aux gastrines/chol6cystokinines chez quelques crustac6s d6capodes: recherche d'un r61e biologique. PhD dissertation, Rennes, 125 p
