Occurrence of analogues of the myotropic neuropeptide orcokinin in the shore crab, Carcinus maenas: Evidence for a novel neuropeptide family

Peptides, Vol. 16, No. 1, pp. 67-72, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0196-978WZ $9.50 + .OO

Pergamon 0196-9781(94)00145-6

Occurrence of Analogues of the Myotropic Neuropeptide Orcokinin in the Shore Crab, CarcLws maenas: Evidence for a Novel Neuropeptide Family DIETER

BUNGART,*

CAROLINE

HILBICH,I_

HEINRICH

DIRCKSEN*

AND

RAINER

KELLER*’

*Institutfir Zoophysiologie der Rheinischen Friedrich Wilhelms-Universitiit, Endenicher Allee 11-13, D-53115 Bonn, Germany and Qentrum fiir Molekulare Biologie, Universitiit Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Received

2 May 1994

BUNGART, D., C. HILBICH, H. DIRCKSEN AND R. KELLER. Occurrence of analogues of the myotropic neuropeptide orcokinin in the shore crab, Cur&us maenas: Evidence for a novel neuropeptide family. PEPTIDES 16(l) 67-72, 1995.-By use of an enzyme immunoassay that was developed for the determination of orcokinin, a myotropic neuropeptide of the sequence NFDEIDRSGFGFN from the crayfish, Orconectes limosus, immunoreactive material was detected in extracts of thoracic ganglia from the shore crab, Curcinlcr muenas. Isolation of the immunoreactive material was achieved by the following steps: I) prepurification by gel filtration, 2) immunoaffinity chromatography on an anti-orcokinin IgG protein-A sepharose column, and 3) reversed-phase HPLC. The HPLC profile after affinity purification revealed three main immunoreactive peptides that were rechromatographed. None of these peptides was identical to orcokinin in terms of retention time. Automated gas-phase sequencing revealed these peptides to be analogues of orcokinin differing in one amino acid residue. They were named [Se?‘]-, [Ala”]- and [VaI’3]orcokinin (NFDEIDRSSFGFN, M, 1549.3; NFDEIDRSGFGFA, M, 1475.3; NFDEIDRSGFGFV, M, 1503.9). Carboxypeptidase A treatment of the peptides indicated a free C-terminus. Complete characterization of the three peptides was achieved from approximately 230 thoracic ganglia of Curcinus maenas. Crustacea Immunoaffinity

Carcinus maenas purification

Myotropic

peptide

Neuropeptide

for reprints

should be addressed

Analogues

determine quantitatively the distribution of orcokinin immunoreactivity (IR) in the nervous system of two crayfish species, Orconectes limosus and Astacus astacus, and the lobster, Homarus americanus (3). In all three species, orcokinin-IR was detected throughout the entire nervous system in a similar distribution. We were interested to determine whether orcokinin or related peptides occur in crustaceans other than the three astacidean species, or perhaps other arthropods or invertebrates. In this article, we describe the isolation and structural elucidation of three orcokinin-related peptides from the nervous system of a brachyuran species, the shore crab, Carcinus maenas. Originally, orcokinin and [Val”]orcokinin were identified by the hindgut bioassay and were isolated by several HPLC purification steps (4,22). In the present study, we developed a more rapid isolation procedure by use of the orcokinin sandwich-ELISA for detection of cross-reactive material (3) and immunoaffinity chromatography for its enrichment. A

RECENTLY, the isolation and complete structural elucidation of orcokinin (NFDEIDRSGFGFN), a myotropic neuropeptide from the abdominal nerve cord of the crayfish, Orconectes limosus, was reported from our laboratory (:!2). Subsequently, an analogue of orcokinin with Val in position 13 instead of Asn was identified in hindgut extracts of the same species (4). Both peptides are highly potent stimulators of crayfish hindgut contractions. Orcokinin and [Val’3]orcokinin join a group of myotropic/ cardioactive neuropeptides in crustaceans that, at present, includes five members of the FMRFamide-related peptides (FaRPs) (10,13,27), proctolin (19,20,26), and crustacean cardioactive peptide (CCAP) (21,24). The latter is as yet the only myotropic neuropeptide that was first identified in crustacean species and later in insects (5,11,23). Our knowledge concerning the occurrence of orcokinin is at present restricted to astacidean crustaceans. The development of a sensitive and highly specific enzyme immunoassay enabled us to

’ Requests

Orcokinin

to Rainer Keller.

67

68

BUNGART

r

80

- 70 m - 60 2 E - 50 4 - 40 x - 30

0

10

20

30

Retention

40

50

60

Tune [min]

FIG. 1. Reversed-phase HPLC of an extract of two thoracic ganglia from Car&us maenas and localization of orcokinin-IR. SepPak (MilliporeWaters)-purified material was applied to a Baker C 18 wide pore column that was equilibrated with 70% solvent A (0.11% TFA in H,O) and 30% solvent B (60% acetonitrile in 0.10% TFA). The peptides were eluted with a linear gradient from 30-80% solvent B in 60 min at a flow rate of 0.9 ml/min. Eluate was automatically collected in 0.9 ml fractions. Note orcokininlike-IR at retention times different from that of orcokinin (arrowhead). Bars represent relative amounts of immunoreactivity.

subsequent two-step HPLC procedure tain the peptides for sequencing.

proved sufficient

to ob-

METHOD

Animals Shore crabs, Carcinus maenas, were collected from the sea shore near Bangor (Gwynedd, UK). They were kept in a recirculating artificial filtered sea water system at 12°C under a 12-h light-dark regime and were fed pelleted cat food.

ET

AL.

pH 7.4) and then measured

photometrically at 225 nm. Orcokinin-immunoreactive (IR) fractions were localized by an enzymelinked immunosorbent assay (ELISA) (3).

ImmunoafJinity Chromatography Anti-orcokinin serum (3) was mixed with protein-A sepharose 4 B fast flow (Sigma), which was preswollen in PBS (pH 7.4). After continuous shaking for 1 h at room temperature (RT), the beads were thoroughly washed with 0.2 h4 sodium borate buffer (pH 9.0). Subsequently, dimethyl pimelimidate as cross-linking agent in a final concentration of 25 mM was added and the mixture was gently shaken for 30 min (7,18). The reaction was stopped by resuspending the centrifuged beads in 0.2 M ethanol amine (pH 8.0) for 2 h under continuous shaking. In a final step, the beads were washed and resuspended in PBS and packed in a small plastic column. Excess, noncross-linked antibodies were eluted with 100 mM glycine/HCl buffer (pH 3.0). Finally, the column was washed with PBS containing 0.02% sodium azide and stored at 4°C. Pooled fractions from the gel filtration, equivalent to batches of a maximum of 60 thoracic ganglia, were applied to the affinity column by means of a peristaltic pump at 4°C. The flow rate was 30 ml/h. Unbound material was removed with PBS. Immunoreactive material was eluted using warm (37°C) 2 N acetic acid and fractionated manually. Orcokinin immunoreactivity of the fractions was determined by ELISA.

High Performance Liquid Chromatography The immunoaffinity-purified material was subjected to reversed-phase HPLC. The chromatographic system consisted of two type 510 pumps, a model 680 solvent programmer, a U6K injector, a model 481 LC spectrophotometer (all Waters Assoc.), and a chart recorder (Perkin-Elmer recorder 56) or, alternatively, an integrator (Waters 740 Data Module). Solvents for gradient 2.0

Tissue Extraction A total of 234 thoracic ganglia were dissected from crabs of both sexes under ice-cold crab saline (14). Ganglia were immediately placed in an extraction medium consisting of 1% formic acid, 5% trifluoroacetic acid (TFA), 1% (w/v) NaCl in 1 N hydrochloric acid (l), and were stored at -20°C until further use. Extraction was carried out by repeated sonication (Branson sonifier) followed by centrifugation for 45 min at 20,000 rpm (2°C) in a Beckman centrifuge (J 21-C; JA 21 rotor). Supematants were removed and stored frozen until further use. Pellets were resuspended in extraction medium, sonicated again, and then stirred overnight at 4°C. After centrifugation, supematants were combined and stored at -20°C.

3

1.5

0 m et E

1.0

3 I? s $

0.5

.

0.0 0

Gel Filtration Gel filtration of thoracic ganglia extracts was performed on Sephadex G-50 (Pharmacia) using 2 N acetic acid as eluent. The bed volume of the column was 145 ml (850 x 17 mm). Prior to gel filtration, samples corresponding to aliquots of 60 thoracic ganglia were concentrated to a volume of approximately 4 ml in a Speed Vat concentrator (Savant, Bachofer). Gel filtration was carried out at a flow rate of 7 ml/h and 150 fractions of 1.5 ml were collected automatically at 4°C. Collected fractions were lyophilized to remove the acetic acid, redissolved in PBS (10 mM Na2HP04.2H20, 1.7 mM KH#O.+, 136 mMNaCl,2.7 mM KCl,

10

20

30

V

40

50

60

Fraction

FIG. 2. Affinity chromatographic purification of orcokinin-IR material. An extract of 30 thoracic ganglia, prepurified by gel filtration and dissolved in PBS (pH 7.4), was applied to the affinity column. Unbound material was removed by washing the column with PBS (fractions l17). The bound fraction was eluted with 2 N acetic acid (37°C) (fractions 18-35 and 41-55). Between the two elution periods, the column was rinsed with PBS (fractions 36-40). Fractions of 1 ml volume were collected manually. Aliquots of the fractions were assayed by ELISA. The eluate was combined and further purified by HPLC (see Fig. 3).

ORCOKININ-LIKE

PEPTIDES

FROM

Carcinus

69

maenas

RESULTS

- 80

,..” _...‘. ,..‘.

- 70

. ...’

_...” . ...’

,....’

,,..”

,_.’

m

-60

2

-50

2 2

- 40

K

- 30

, 0

c

10

I

20 Retention

I

30 Time

c

40

I

50

1

60

[min]

FIG. 3. HPLC of the immunoaffinity purified material (Fig. 2) from Curcinus maenas. Chromatography was carried out with a linear gradient from 30-80% solvent B in 60 min on a PBondapak phenyl column (Millipore- Waters). The flow rate was 0.9 ml/min. Immunoreactive peaks were localized by ELISA (asteriks). The three main immunoreactive peaks, marked as A, B, and C, were rechromatographed (see Figs. 4, 5,6). None of these peaks corresponded to the retention time of orcokinin (arrowhead).

elution were 0.11% TFA in H20 (A) and 60% acetonitrile in 0.10% TFA (B). (For details concerning columns and chromatographic conditions see legends to figures.)

Amino Acid Analysis. Sequence Analysis, and FAB-Mass Spectrometry

Extracts from the nervous system of the shore crab, Cur&us muenas, tested positively in the ELISA that was developed for the determination of orcokinin. However, the cross-reactivity was quite low, and increasing extract concentrations gave a rather flat curve that was unsuitable for a precise quantification of the immunoreactive material as orcokinin equivalents. The test nevertheless proved useful because the qualititative or, at best, semiquantitative response was sufficient to monitor orcokinin-like immunoreactive material during the isolation procedure. Figure 1 shows the separation of SepPak-prepuriiied thoracic ganglia extracts by HPLC and the results of the ELISA. Orcokinin-IR was detectable in several fractions, especially in fractions 18-28, which indicated the presence of at least three immunoreactive peptides. Additionally, somewhat lower immunoreactivity was measured in fractions 12 and 40-41. For isolation of the orcokinin-IR material a total of 234 thoracic ganglia were extracted. The extract was prepurified by gel filtration on Sephadex G-50, to remove proteins and salts from the acidic extracts. Orcokinin-IR was associated exclusively with the fractions that eluted between the protein peak and the salt peak (not shown). AI1 fractions corresponding to this peptide peak region were pooled and the material was subjected to affinity chromatographic purification, which resulted in a considerable enrichment of immunoreactive material (Fig. 2). Eluted fractions 18-35 and 41-55 were pooled, Speed Vat concentrated, and subjected to HPLC analysis, first on a PBondapak phenyl column (Fig. 3). This separation gave three main peaks marked A, B, and C. Some smaller peaks also showed immunoreactivity in the ELISA, but quantities were considered to be too small for further purification and they did not appear reproducibly in all runs. None of the immunoreactive peptides seemed to be authentic orcokinin because they displayed different retention times. For example, cochromatography of a mixture of peptide A (which

Samples of purified peptides (approximately 50 pmol) were hydrolyzed in vacua in 30 111constant boiling HCl (Sigma) for 1 h at 150°C. Amino acid analysis was performed on a conventional analyzer (Biotronic LC 5000) operating with orthophthaldialdehyde postcolumn derivatization (2). Automated sequence analysis was performed on a gas-phase sequencer (Applied Biosystems, 477 A), on-line connected to a phenylthiohydantoin analyzer (Applied Biosystems, 120 A). Peptide samples of at least 100 pmol were analyzed. FAB-mass spectrometry of peptide samples, amounting to approximately 10 pmol, was performed on a Siex API-III mass spectrometer (6). Carboxypeptidase

A Treatment

Samples of approximately 100 pmol of purified peptide were dried in a Speed Vat concentrator (Savant, Bachofer) and redissolved in 50 ~1 of 20 mM triethanolamine hydrochloride (pH 7.5), containing 200 mM NaCI. Two units of carboxypeptidase A (Boehringer) in 23 ~1 of the same buffer were added and incubated for 1 h at 25°C. After addition of 100 ~1 of a 5% aqueous TFA solution, the mixtures were subjected to HPLC analysis. Enzyme-Linked

Immunosorbent

Assay (ELISA)

The ELISA for orcokinin, which is a noncompetetive, indirect sandwich-type assay, has b,een described in detail in a previous article (3). In this present study, the original procedure was followed without modification,

Retention FIG.

Time

[min]

4. Two successive rechromatographies of peak A (Fig. 3) on a Baker Cl8 wide pore column (a). Elution: Linear gradient from 30-60% solvent B in 50 min. Flow rate: 0.9 ml/min. Purity of peptide A was checked in a second rechromatography under the same conditions (b).

70

BUNGART ET AL.

eluted closest to the retention time of synthetic orcokinin) and orcokinin resulted in two peaks (not shown). Immunoreactive peptides A, B, and C were further purified on a C 18 column [Figs. 4, 5, 6(a)], which showed that high purity of the peptides was already accomplished during the first HPLC step. Prior to amino acid analysis and sequence analysis, the purity of the peptides was checked in a second rechromatography on a Cl8 column [Figs. 4, 5, 6(b)]. From a total of 234 thoracic ganglia we obtained approximately 1 nmol of peptide A, 2 nmol of peptide B, and 500 pmol of peptide C after the second HPLC step. Aliquots of approximately 50 pmol were subjected to amino acid analysis. The amino acid composition of the three peptides is shown in Table 1. Impurities in the blank contributed to increased values for Glx (Gln, Glu) and Gly, which would lead to overestimation of these amino acids. However, amino acid analysis data clearly suggested that the purified peptides A, B, and C were orcokinin related, obviously with amino acid substitutions in one position. In peptide A, one of the Gly residues of orcokinin seemed to be replaced by Ser, and in peptides B and C, one Asx (Asn or Asp) seemed to be replaced by Ala or Val, respectively. Automated gas-phase sequencing of peptide samples of at least 100 pmol unequivocally revealed the complete primary structures. In peptide A, Gly’ is replaced by Ser, and in peptides B and C, As@ is replaced by Ala and Val, respectively. Molecular mass determination of the peptides by FAB-mass spectrometry confirmed these data, but it was not deducible whether the C-terminal amino acid residue was free or amidated. Amino acid sequences and molecular weights of these analogues of orcokinin are listed in Table 2. They were named [Sefiorcokinin, [Ala”]orcokinin, and [Val”]orcokinin. To test whether the C-terminus of the analogues is free or blocked, we incubated samples of peptides A, B, C (100 pmol each) with carboxypeptidase A. HPLC separation of the mixtures after carboxypeptidase A treatment showed complete degradation of the peptides, pointing to a free C-terminus (data not 1

r

_::.I b

0

10

20

30

40

50

Retention Time [min] FIG. 5. Two successive rechromatographies of peak B from Fig. 3 on a Baker Cl8 wide pore column (a). Gradient: 35-70% solvent B in 50 min. The second rechromatography of peptide B was performed under the same conditions (b).

b

0

10

20

30

40

50

Retention Time [min] FIG. 6. First (a) and second (b) rechromatography of immunoreactive material of peak C from Fig. 3. Chromatographic conditions were the same as in Fig. 5.

shown). For comparison, a synthetic C-terminally amidated analogue of orcokinin remained unaffected. DISCUSSION

Following the isolation and characterization of orcokinin, a novel myotropic neuropeptide from the crayfish Orconectes li~OSUS (3,22), we were interested in investigating how widely this peptide or related forms are distributed among crustaceans or other invertebrates. In a brachyuran species, the shore crab Carcinus maenas, ELISA determinations suggested the presence of orcokinin-IR material. Obviously, this material was different from orcokinin because, although it tested unambiguously positive, increasing concentrations gave a flat, irregular curve that did not permit precise quantification. The sandwich-ELISA has been shown to be highly specific for orcokinin, and recent studies on synthetic analogues of orcokinin demonstrated that slight changes of the molecule, especially at the C-terminus, led to a sharp decrease of binding and resulted in irregular binding curves (e.g., a decrease of O.D. values at higher concentrations) (3). Even the heretofore only known natural analogue of orcokinin, [Val13]orcokinin (4), did not readily cross-react in the ELISA. Localization of immunoreactivity after HPLC separation of a thoracic ganglia extract suggested the occurrence of at least three orcokinin-IR peptides in the crab. Immunoaffinity chromatography allowed a rapid and specific enrichment of orcokinin-IR peptides. Following this prepurification step, two successive HPLC runs were sufficient to obtain three orcokinin-IR peptides in pure form for amino acid analysis, sequence analysis, and FAB-mass spectrometry. As predicted from the ELISA and the HPLC retention times, none of these peptides was orcokinin. But they were identified as analogues of orcokinin with one amino acid substituted in each. One of these analogues, [Val’3]orcokinin, was already known from the crayfish, Orconectes limosus (4),

ORCOKININ-LIKE

71

PEPTIDES FROM Curcinus maenas

TABLE 1 AMINO ACID ANALYSIS OF ORCOKININ-IR PEPTIDES A, B, AND C

pm01

Amino acid Asx (Asn, Asp) Ser Glx (Gin, Glu) GUY Ala Val

Number

pmol

Number

3 1

79.94 31.50 39.28 72.57 29.37

3 1 1 2

-

137.40 48.82 75.74 119.65 51.79 -

1 2-3 1

46.08 132.76 49.94

3-4 2

I 1

27.39 74.:32 30.63

Ile Phe Arg

pm01

Number

105.‘11 48.55 43.49 46.24 -

Peptide C

Peptide B

Peptide A

I 2 1 -

1

4 1 1 2 -

1 3 1

1 3 1

-

28.56 81.36 29.79

1 3 1

orcokinin Number

Numbers of amino acids are relative to Arg.

Concerning the biological effects of the ORPs in Car&us our investigations are still at a very preliminary stage. First experiments showed that they have no noticeable stimulatory activity on the heart as tested in an in situ preparation. However, in the crayfish, Orconectes limosus, the heart-accelerating effect was also very low, in contrast to the very potent action on the hindgut. We have already proposed that the heart is uniikely to be a major target organ of orcokinin. The hindgut of the crab, when connected to a force transducer, is not spontaneously active and does not readily respond to myotropic peptides. In preliminary experiments, however, we found that electrically induced contractions (0.166 Hz, 5 V) are significantly potentiated by 1 X lo-’ M [Val’3]orcokinin. Clearly, more work needs to be done on the physiological significance of the ORPs in Carcinus. Some clues may come from immunocytochemical mapping of ORPs in the nervous system, which are in preparation. Including the presently known four ORPs, the list of myotropic/cardioactive peptides known from crustaceans is now extended to 11. As mentioned in the introduction, the occurrence of proctolin, CCAP, and the FaRPs is not restricted to crustaceans. Proctolin and CCAP are well established as insect and crustacean neuropeptides [for reviews see (8,9,15)], and members of the FaRPs were additionally identified in several other invertebrate taxa (e.g., molluscs, coelenterates, and nematodes), and even in vertebrates [for review see (17)]. In this context, it is of interest to investigate the distribution of the ORPs. The orcokinin ELISA and in particular the immunoaffinity purification, as described in this study, should prove to be useful tools for further studies on the distribution of the

and probably occurs in other astacidean crustaceans. The distribution of this peptide in the nervous system of astacidean crustaceans in relation to orcokinin would be of particular interest for further investigations. At present, we know four orcokinin-related peptides (ORPs) from two decapod crustacean species. This establishes a novel neuropeptide family. A characteristic feature of the ORPs is the lack of both Nand C-terminal blocking groups, which is relatively untypical for neuropeptides but not without example. The myotropic peptide proctolin, the enkephalins, for which presence in Carcinus was recently ascertained (12) and LOM-AG-myotropin, a myotropic peptide of the male accessory reproductive glands of Locusta migratoria (16), are also unblocked peptides. For proctolin, the C-terminal hydroxyl-group is a necessary structural requirement for its bio’logical function (25), whereas our structure-activity studies on orcokinin showed that the C-terminal hydroxyl-group is not of substantial significance for its biological activity (Bungart et al., submitted). It may be speculated that the free C-terminus is related to a short-term action of these peptides, which may be correlated with a rapid inactivation. The problem has been discussed in some detail in a previous article (3). The differences between the presently known four ORPs are limited to one amino acid residue. Interestingly, the substitutions are all found in positions 9 and 13. The latter finding agrees well with results from structure-activity studies showing that C-terminal changes (deletions md amidation) reduce the biological activity in the crayfish hindgut assay less than N-terminal modifications. Orcokinin and [Val”]orcokinin proved to be equally potent in the assay (Bungart et al., submitted).

mamas,

TABLE 2 RESULTS OF SEQUENCE ANALYSIS

AND FAB-MASS

SPECTROMETRY

OF ORCOKININ-RELATED

PEFWDES

M (Da)

Sequence

(A) [SeP]OK: (B) [Ala’3]0K: (C) [Val’3]OK: Orcokinin (OK):

Measured CalculaM

Asn Asn Asn Asn

Phe Phe l?he Phe

Asp Asp Asp Asp

Ghl Glu Glu Glu

Ile Ile Ile Ile

Asp ASP Asp Asp

Arg Arg Arg Arg

Ser Ser Ser Ser

Ser GlY GUY GUY

Phe Phe Phe Phe

GUY GUY GUY GlY

Phe Phe Phe Phe

Asn

Ala Val Asn

1549.3 1475.3 1503.9 1519.7

1547.6 1474.6 1503.6 1517.5

72

BUNGART ET AL.

ORPs among crustaceans, arthropods, and possibly other invertebrate groups, and for the identification of additional analogues of this neuropeptide family. Our ELISA studies on brain extracts of the locust, Locusta migratoria, yielded preliminary evidence that orcokinin-IR peptides may occur in this species.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Deutsche Forschunggemeinschaft (Ke 206/7-9) to R.K. We are grateful to Prof. K. Beyreuther (ZMBH, Heidelberg) for making his sequencing facilities available. Thanks are also due to Dr. A. Saiiter (Bonn) for help with the FAB-mass spectrometry.

REFERENCES 1.

2. 3.

4.

5. 6. 7. 8.

9. IO. 11. 12.

13. 14.

Bennett, H. P. J. Peptide extraction methods. In: Gross, B.; Brown, C. A.; Goltzmann, D.; Solomon, S., eds. Peptides: Structure and biological functions. Rockford, IL: Pierce; 1979: 121- 13 1. Benson, J. R.; Louie, R.; Bradshaw, R. A. Amino acid analysis of pep tides. In: Gross, E.; Meienhofer, J., eds. The peptides. Anal&is, sy~&sis. bioloev. vol. 4. New York: Academic Press: 1981:217-260. B;ngart,%.; Dircksen, H.; Keller, R. Quantitative determination and distribution of the myotropic neuropeptide orcokinin in the nervous system of astacidean crustaceans. Peptides 15:393-400; 1994. Burdzik, S.; Sauter, A.; Stangier. J.; Keller, R. A novel orcokininrelated myotropic peptide from the hindgut of the crayfish, Orconecks limosus. In: Elsner, N.; Heisenberg, M., eds. Gene-brainbehaviour. Stuttgart: Thieme; 1993598. Cheung, C. C.; Loi, P. K.; Sylwester, A. W.; Lee, T. D.; Tublitz, N. J. Primary structure of a cardioactive neuropeptide from the tobacco hawkmoth, Manduca sexra. FEBS 3 13: l65- 168; 1992. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, F. F.; Whitehouse, C. M. Electrospray analyzation for mass-spectrometry of large biomolecules. Science 246:64-71; 1989. Gersten, D. M.; Marchalonis, J. J. A rapid novel method for the solid-phase derivatization of IgG antibodies for immunoaffinity chromatography. J. Immunol. Methods 44:305-309; 1978. Holman, G. M.; Nachman, R. J.; Wright, M.; Schoofs, L.; Hayes, T. K.; De Loof, A. Insect myotropic peptides: Isolation, structural characterization and biological activities. In: Menn, J. J.; Kelly, T. J.; Masler, E. P., eds. Insect neuropeptides: Chemistry, biology and action. Washington, DC: American Chemical Society; 199 l:4050. Keller, R. Crustacean neuropeptides: Structures, functions and comparative aspects. Experientia 48:439-448; 1992. Krajniak, K. G. The identification and structure-activity relations of a cardioactive FMRFamide related peptide from the blue crab Callinectes sapidw Peptides 12: I295- 1302; 1991. Lehman, H. K.; Mugiuc, C. M.; Miller, T. A.; Lee, T. D.; Hildebrand, J. G. Crustacean cardioactive peptide in the sphinx moth, Manduca sexta. Peptides 14735-741: 1993. Liischen, W.; Buck, F.; Willig, A.; Jaros, P. P. Isolation, sequence analysis, and physiological properties of the enkephalins in the nervous tissue of the shore crab Carcinus maenas L. Proc. Natl. Acad. Sci. USA 88:8671-8675; 1991. Mercier, A. J.; Orchard, I.; Te Brugge, V.; Skerrett, M. Isolation of 2 FMRFamide related peptides from crayfish pericardial organs. Peptides 14:131-135; 1993. Nordmann, J. J.; Morris, J. F. Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab. Cell Tissue Res. 205:31-42; 1980.

15. Orchard, I.; Belanger, J. H.; Lange, A. B. Proctolin: A review with emphasis on insects. J. Neurobiol. 20:234-254, 1989. 16. Paemen, L.; Schoofs, L.; Proost, P.; Decock, B.; De Loof, A. Isolation, identification and synthesis of LOM-AG-Myotropin II, a novel peptide in the male accessory reproductive glands of Locusta migratoria. Insect Biochem. 21:243-248; 1991. 17. Price, D. A.; Greenberg, M. J. The hunting of the FaRPs: The distribution of FMRFamide-related peptides. Biol. Bull. 177: 198-205; 1989. 18. Schneider, C.; Newman, R. A.; Sutherland, D. R.; Asser, U.; Greaves, M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem. 257: 10766- 10769; 1982. 19. Schwarz, T. L.; Lee, G. M.; Siwicki, K.; Standaert, D. G.; Kravitz, E. A. Proctolin in the lobster: The distribution, release and chemical characterization of a likely neurohormone. J. Neurosci. 4:13001311; 1984. 20. Stangier, J.; Dircksen, H.; Keller, R. Identification and immunocytochemical localization of proctolin in pericardial organs of the shore crab, Carcinus maenas. Peptides 7167-72; 1986. 21. Stangier, J.; Hilbich, C.; Beyreuther, K.; Keller, R. Unusual cardioactive peptide (CCAP) from the pericardial organs of the shore crab, Carcinus maenas. Proc. Natl. Acad. Sci. USA 84:575-579; 1987. 22. Stangier, J.; Hilbich, C.; Burdzik, S.; Keller, R. Orcokinin: A novel myotropic peptide from the nervous system of the crayfish, Orconectes limosus. Peptides 13:859-864; 1992. 23 Stangier, J.; Hilbich, C.; Keller, R. Occurrence of crustacean cardioactive peptide (CCAP) in the nervous system of an insect, Locusta migratoria. J. Comp. Physiol. [B] 159:5-l 1; 1989. 24. Stangier, J.; Keller, R. Occurrence of the crustacean cardioactive peptide (CCAP) in the nervous system of the crayfish, Orconectes limosus. In: Wiese, K.; Krenz, W. D.; Tautz, J.; Reichert, H.; Mulloney, B., eds. Frontiers in crustacean neurobiology. Basel: Birkhguser; 1990:394-399. 25. Starratt, A. N.; Brown, B. E. Analogs of the insect myotropic peptide proctolin: Synthesis and structure-activity studies. Biochem. Biophys. Res. Commun. 90: 1l25- 1130;1979. 26. Sullivan, R. E. A proctolin-like peptide in crab pericardial organs. J. Exp. Zool. 210:543-552; 1979. 27. Trimmer, B. A.; Kobierski, C. A.; Kravitz, E. A. Purification and characterization of immunoreactive substances from the lobster nervous system: Isolation and sequence analysis of two closely related peptides. J. Comp. Neurol. 266:16-26; 1987.