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Characterization of a second peptide with adipokinetic and red pigment-concentrating activity from the locust corpora cardiaca
Insect Biochem., Vol. 9, pp. 497 to 501. © Pergamon Press Ltd. 1979. Printed in Great Britain.
0020-1700/79/0901-0497 $02.00/0
CHARACTERIZATION OF A SECOND PEPTIDE WITH ADIPOKINETIC AND RED PIGMENT-CONCENTRATING ACTIVITY FROM THE LOCUST CORPORA CARDIACA JENS CARLSEN,* WILLIAM S. HERMAN,t MOGENS CHRISTENSEN ++a n d EARS JOSEFSSON~ *Department of Biochemistry C, University of Copenhagen, Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark "tDepartment of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108, U.S.A. ++Novo Research Institute, Novo Alle, DK-2880 Bagsv~erd, Denmark (Received 4 December 1978) A new peptide with both adipokinetic activity in the locust and red pigment-concentrating activity in the shrimp, can be readily separated from the adipokinetic hormone in extracts of corpora cardiaca (CC) from Sehistoeerca americana gregaria by the use of gel chromatography and the Leander adspersus bioassay. The new peptide accounts for 20~, of the total biological activity in locust CC, and it is located principally in the CC glandular lobe. The amino acid composition of the pure peptide is: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. This composition is similar to, but not identical with the red pigmentconcentrating hormone of the shrimp. Key Word Index: Invertebrate peptide hormones, locust neurosecretory hormones, adipokinetic hormones in locust Abstract
INTRODUCTION
Hormone assays
LOCUST c o r p o r a cardiaca (CC) contain an adipokinetic h o r m o n e ( A K H ) that has been purified, characterized, a n d synthesized (STONE et al., 1976; BROOMFtELD and HARDY, 1977). Extracts from locust CC, as well as synthetic A K H also show hyperglycemic activity in cockroaches a n d red pigment-concentrating activity in crustaceans (MoRDUE and GOLDSWORTHY, 1969; MORDUE and STONE, 1976, 1977; THOMSEN, 1946). The possible existence in the locust C C of additional substances with the biological actions of synthetic A K H has been suggested on the basis of several experiments (MORDUE a n d GOLDSWORTHY, 1969; G,~DE a n d HOLWERDA, 1976), but attempts to separate a n d characterize such c o m p o u n d s have to date been unsuccessful (HOLWERDA et al., 1977). Using the highly sensitive, rapid, and convenient Leander adspersus e r y t h r o p h o r e bioassay (FERNLUND, 1968), we have t a k e n a d v a n t a g e of the red pigmentc o n c e n t r a t i n g activity o f A K H - l i k e substances (HERMAN et al., 1977) to isolate a n d characterize a second peptide with the biological effects of A K H from locust CC. O u r results are described below. MATERIALS AND METHODS
Adipokinetie response. Total haemolymph lipid was measured by the vanillin method (GOLDSWORTHV et al., 1972). Haemolymph (10 #1) was taken from a puncture in the membrane at the base of the hind leg. Sampling was followed by injection of a 50 #1 aliquot of the solution to be tested into the abdomen. After 1 hr a second 10 ~1 haemolymph sample was taken. Samples were immediately mixed with 0.4 ml concentrated sulphuric acid, and the mixtures were stirred thoroughly and analyzed for lipid content. Red pigment-concentrating activity. This was assayed on eyestalkless L. adsperus using a (2+2) point parallel line assay with ten animals at each dose (F~RNLUNO, 1968). Synthetic red pigment-concentrating hormone (RPCH) dissolved in 1.6~o (w/v) NaC1 was used as a standard. Concentrations of the standards were 0.47 × 10- ~o M and 1.41 × 10 ~0 M, respectively. Standards or samples (50 ill) to be tested were injected into each animal. A simplified procedure was used when assaying the separate tube fractions from column effluents and electrophoresis extracts for their activity. Samples of each fraction were appropriately diluted in 1.6",, (w/v) NaCI and 50/fl was injected into each of two animals at only one dose for each fraction. Activity responses were recorded as in the complete assay procedure. As no standard was included, no attempt was made to evaluate the quantitative amount of hormone in the different tube fractions of the column chromatographic separations. Thus, effluent curves show only the averaged responses for each tested fraction.
Experimental animals
Chemicals
Schistocerca americana gregaria were reared under crowded conditions with a 16 hr daily photo-period and a temperature of about 30°C. They were fed with bran, fresh grass and lettuce. Leander adspersus caught at the Danish coast were kept in l-m 3 closed system marine aquaria at 10°C. CC from freshly killed adult locusts were obtained by dissection. The locust Were decapitated and dissections carried out under chilled saline (0.128 M NaCI + 0.005 M KCI). The glands were stored at -20°C until extraction.
Sephadex G-25 fine and Sephadex LH-20 were purchased from Pharmacia, Uppsala, Sweden. Haemolymph lipid concentrations were determined using a Total Lipids kit obtained from Boehringer. All chemcials were of analytical grade. Water was delivered from a Milli-Q system, M illipore. Synthetic RPCH (FERNLUND and JOSEFSSON, 1972) was prepared in our laboratory (CHRISTENSEN, CARLSEN and JOSEFSSON, 1979) and had the same specific activity as the native hormone purified from L. adspersus (CARLSENel al., 1976). 497
JENS CARLSENet al.
498
Thin-layer chromatography Thin-layer chromatography (TLC) was performed on precoated TLC plates, Silica Gel 60 F-254 from E. Merck. Chromatograms were visualized by spraying with tert-butyl hypochlorite followed by p-tolidine/potassium iodide or with 10% (w/v) 4-N,N-dimethylamino-benzaldehyde in concentrated HCI, mixed with acetone (1:4, v/v) prior to use (Ehrlich reagent). The solvent systems used were; S-I; 2propanol-concentrated ammonia-water (8:1:1, by vol), S-2; ethyl aceta te-ethanol-water-pyridine-acetic acid (60:30:8:2:1, by vol), S-3; 2-propanol-water-acetic acid (25:10:1, by vol).
Amino acM analysis A Kontron Liquimat III, Kontron International Ziirich was used. Samples were hydrolized in sealed ampoules at I10°C for 24 hr in 6 M HCI. For determination of tryptophan, samples were hydrolyzed in 3 N mercaptoethane sulphonic acid (PENKEet al., 1974) and analyzed on a Beckmann Multichrom Liquid Column Chromatograph 4255.
Electrophoresis Glucose and alanine (serving as controls for the electrophoresis) were added and the samples applied on paper (Whatman No. 1). Electrophoresis was conducted at pH 2.3 [10% (v/v) acetic acid] and pH 6.0 (pyridine-acetic acid-water, 9:1:90, by vol) for 2 hr at 250 V. RESULTS
Preparation
of aqueous extract
CC were homogenized with ice-cold water (0.1 ml/CC) for 2 min in an all-glass Potter-Elvehjelm homogenizer, and the resulting suspension boiled for 5 min. After chilling on ice the suspension was centrifuged at 17,000 g for 15 min at 4°C. The supernatant was collected and the sediment extracted with a volume of water corresponding to 1/5 of the supernatant. The combined supernatants were frozen and thawed, and the small sediment formed removed by centrifugation. The resulting solution was lyophilized and 80 #g/CC of dry, pale yellow material obtained. The powder was extracted with water (0.4
ml/mg) at room temperature for 30 min. The insoluble material was centrifuged down (17,000 g, 15 min at 4°C) and the supernatant collected. The sediment was washed twice with a volume of water corresponding to 1/5 of the supernatant, before being discarded. The supernatants were combined: 1 ml of this aqueous extract corresponded to approx 25 CC.
Localization o f red pigment-concentrating activity and adipokinetic activity in gel filtration As shown in Fig. 1 red pigment-concentrating activity and adipokinetic activity are eluted over the same volume range from a G-25 column. The sensitive L. adspersus assay revealed that the red pigmentconcentrating activity could be separated; of the two distinct activities, peak I contained five times more activity than peak II. The same fractions also showed adipokinetic activity but two peaks could not be clearly demonstrated, probably on account of the reported limitations of the lipid mobilisation assay (HoLWERDA et al., 1977). Extracts prepared by ultrasonic treatment of the tissue in methanol according to SXONE et al. (1976) shows the same red pigment-concentrating activity 'pattern after gel filtration as the aqueous extract. In one experiment an aqueous extract prepared from Locusta migratoria C C was chromatographed on Sephadex G-25, and the same red pigmentconcentrating activity profile as described from S. americana gregaria CC was obtained. The localization of the red pigment-concentrating activity in separated glandular and storage lobes was investigated. Glandular lobes contain ten times more total activity than the corresponding storage lobes, but extracts from either part of the gland showed the same distribution between peak I and peak II as extracts from whole glands.
Preliminary characterization oJ the red pigmentconcentrating activities The aqueous extract (2 ml) with a red pigmentconcentrating activity corresponding to 2.0 #g R P C H
E
f~D 0 o4
.~
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o
o
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' EFFLUENT(mll
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E
...A
o
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Fig. 1 Gel filtration on Sephadex G-25 of the aqueous extract of corpora cardiaca. The extract (1 ml) was applied to an 1.4 cm i.d. x 36.0 cm column and eluted with water at 24°C. Fractions of 1.5 ml were collected and assayed. ( © - - © ) Red pigment-concentrating activity of the fractions assayed in L. adspersus at dilutions of 1:100. Adipokinetic activity assayed in S. americana gregaria with undiluted fractions. ( - - ) . Transmittance at 206 nm.
Locust corpora cardiaca peptides
499
-15 hJ tt') Z
-10 o -5
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O
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EFFLUENT(ml)
Fig. 2 Chromatography on LH-20 Sephadex. Column: 1.3 cm i.d. x 37.0 cm, eluted with 6'I. (v/vj lbutanol. A. Elution profile of compound I, fractions of 1.5 ml were collected, diluted 1:600and assayed for red pigment-concentrating activity. B. Elution profile of compound II, fractions of 1.5 ml were collected, diluted 1:100 and assayed for red pigment-concentrating activity.
was applied to a G-25 Sephadex column (2.5 cm i.d. x Purification oJ the active substances 92.5 cm) and eluted with water. Fractions of 8.4 ml Purification of the two substances with red pigmentwere collected. The two distinct red pigment- concentrating activity was achieved from 37 ml concentrating activities were completely separated; aqueous extract (corresponding to 920 CC) using the compound 1 eluted at 0.9 × V, (column volume) and procedure described in the preliminary charactericompound II, like RPCH (FERNLUNDand JOSEFSSON, zation. The column dimensions were: Sephadex G-25; 1968) retarded on the gel, eluted at 1.2 x Vt. The 5.0 cm i.d. × 89.0 cm and Sephadex LH-20; 2.5 cm i.d. active fractions in the two peaks were combined, and x 87.0 cm. Special care was taken to avoid evaporated to dryness under reduced pressure. The contamination from dust, etc., and the demineralized small residues were redissolved in 6~o (v/v) 1-butanol water was glass-distilled prior to use. After the final gel and subsequently chromatographed on a Sephadex filtration all solvent was evaporated and the residues LH-20 column eluted with 6% (v/v) 1-butanol. In Fig. taken in solution in a small volume of methanol 2 the elution profile of the two red pigment- (compond l: 1000 /A, compound II: 400 /A) and concentrating activities is shown. Compound I was analyzed. only slightly retarded on the gel and was eluted at 1.1 × Vt whereas compound II again behaved like RPCH Analysis of compound I and lI The methanolic solutions of the two isolated and (FERNLUND and JOSEFSSON, 1968) and was strongly retarded (elution vol: 1.8 × V,) in this purified compounds were examined by thin-layer chromatographic system. The combined active chromatography (reference: 1 #g synthetic RPCH) fractions (9 ml) after Sephadex LH-20 chromatog- and they were homogenous in all three media tested raphy of compound I, contained red pigment- (Table 1). The spots could also be developed spraying concentrating activity corresponding to 1.0 pg RPCH with Ehrlich reagent, indicating that the two peptides and 2 /~1 of the combined fractions increased the contain tryptophan. Amino acid analysis after acid haemolymph lipid concentration 14.8 _+ 0.9 mg/ml hydrolysis showed that compound I had the amino (mean _+ S.E.M., n = 6 ) when injected into S. acid composition: Asp2, Thr z, Glu, Pro, Gly, Leu, americana gregaria. The combined active fractions (13 Phe, and compound II the composition: Asp, Thr, Ser, ml) after Sephadex LH-20 chromatography of Glu, Gly, Leu, Phe (Table 2). Compound I (27 nmole) compound II contained red pigment-concentrating and compound II (25 nmole) (calculated from the amino activity corresponding to 0.2/~g RPCH and 10 pl of acid content) were obtained from 920 CC. Amino acid the combined fractions increased the haemolymph analysis, after hydrolysis in mercaptoethane sulphonic lipid concentration 8.1 + 1.5 mg/ml (mean _+ S.E.M., acid of a small amount (about 3 nmole) of the two peptides n = 6 ) when injected into S. americana gregaria. showed that tryptophan occurred in the same molar No difference was observed between compound I ratio as leucine. Quantitative assay of the purified and compound II on electrophoresis. When the papers peptides showed that they both are 1/50 as active as after electrophoresis at both pH values were cut in 0.5 cm RPCH when injected in L. adspersus, i.e. doses at pieces, extracted in 1.6% (w/v) NaCI and assayed for l x 10 -13 mole cause a significant response. Log red pigment-concentratingactivity, only extracts from dose-response curves were parallel with those pieces cut at the origin showed activity. obtained for RPCH.
500
JENS CARLSENet al.
Table 1. Rs values of compound I, compound I1 and RPCH. Approximately 1 #g each of the isolated hormones and the synthetic RPCH were spotted on each plate Solvent system S-1 S-2 S-3 Compound I Compound lI
0.37 0.42
0.14 0.34
0.63 0.69
RPCH
0.46
0.25
0.66
Table 2. Amino acid composition of the purified peptides. 100 pl of the methanolic solution of compound I and 200/~1of the methanolic solution of compound II were hydrolyzed Compound I nmole* Ratio Asp Thr Ser Glt~ Pro Gly Ala Cys Val Met lieu Leu Tyr Phe Lys His Arg
54.1 53.2 n.d. 26.1 25.1 31.4 n.d. n.d. n.d. n.d. n.d. 27.9 0.6 27.7 n.d. n.d. n.d.
Compound II nmole* Ratio
1.94 1.91 0.94 0.90 1.13 ----1.00 0.02 0.99
12.6 11.3 9.9 12.2 n.d. 12.9 n.d. n.d. n.d. 0.4 n.d. 12.2 n.d. 12.0 n.d. n.d. n.d.
1.03 0.93 0.81 1.00 -1.06 --0.03 -1.00 -0.98 ----
* n.d.: not detectable.
DISCUSSION Our demonstration that the locust corpus cardiacum contains two different biologically active peptides agrees with the early observation of MORDUE and GOLDSWORTHY (1969). Also chromatograms published by HOLWERDAet al. (1977), of extracts from Loeusta migratoria corpora cardiaca on Biogel P-2 show that the adipokinetic activity is eluted over a wide volume range, indicating lack of homogeneity. O f the two adipokinetic active peptides isolated, compound I has an amino acid composition identical with the composition of the A K H purified from S. americana gregaria and L. migratoria CC glandular lobes by STONE et al. (1976). This hormone is a decapeptide with the following structure: pGlu-LeuAsn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH 2 confirmed by chemical synthesis (BROOMFIELD and HARDY, 1977). C o m p o u n d II is probably an octapeptide with the following amino acids in equimolar amounts: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. The tryptophan content is demonstrated by the positive reaction with Ehrlich reagent on the T L C plates, the presence of tryptophan in amino acid analysis after hydrolysis in 2-mercaptoethane sulphonic acid, and by the red
pigment-concentrating activity since tryptophan is indispensible for the red pigment-concentrating activity (JOSEFSSON et al., 1977, CHRISTENSEN et al., 1978). The composition of compound Il is almost identical with RPCH: pGlu-Leu-Asn-Phe-Ser-ProGIy-Trp-NH 2 (FERNLUND and JOSEFSSON, 1972), and shows the same characteristic behaviour when chromatographed on Sephadex gels, indicating a structure very similar to RPCH. C o m p o u n d II like A K H and RPCH, is electrophoretically immobile at neutral and acid pH. These data prove that, like A K H and RPCH, compound II has blocked NH2-terminus and no free carboxyl groups, i.e. a blocked C O O H terminus and the aspartic acid residue as its amide. C o m p o u n d II in the present study has the same red pigment-concentrating activity as A K H (compound I) in L. adspersus. C o m p o u n d II also has, estimated from the relation between the lipid mobilization effect and the red pigment-concentrating activity, about the same adipokinetic activity as A K H in S. americana gregaria. The amount of compound II/CC, calculated from the distribution in the Sephadex G-25 gel filtration, is 1/5 of A K H , but the yield in the purification is lower. C o m p o u n d 11 is located in both the glandular and the storage lobes but occurs as A K H mainly in the glandular lobe. Owing to the minimal amount of material available (about 0.03 #g pure peptide was obtained from one S. americana gregaria), it was not possible in this study to elucidate the structure of compound II. But using the purification system described, A K H is easily separated, so purification of amounts allowing for structural analysis should be possible. This study also opens up for investigation the possible physiological role of this second peptide with adipokinetic activity in the locust neuroendocrine system. Acknowledgement~We thank Ms B. HARDERfor skillful technical assistance, and E. LUNDfor the amino acid analysis. The investigation was aided by grants from the Danish Natural Science Research Council (Project No. 3565) and the University of Minnesota Graduate School. USPHS grant HD-07336. REFERENCES BROOMFIELDC. E. and HARDYP. M. (1977) The synthesis of locust adipokinetic hormone. Tetrahedron Lett. 25, 2201-2204. CARLSEN J., CHR1STENSEN M. and JOSEFSSON L. (1976) Purification and chemical structure of the red pigmentconcentrating hormone of the prawn Leander ad6lwrsus. Gen. comp. Endocr. 30, 327-331. CHRISTENSEN M., CARLSEN J. and JOSEFSSON L. (1978) Structure-function studies on red pigment-concentrating hormone; The significance of the terminal rc~Jdues. HoppeSeyler's Z. physiol. (;hem. 359, 813-818. CHRISTENSEN M., CARLSENJ. and JOSEFSSONL, Structurefunction studies on red pigment-concentrating hormone. I1. The significance of the C-terminal tryptophan amide. Hoppe-Seyler's Z. physiol. Chem. (In press). FERNLUNDP. (1968) Chromactivating hormones of Pandalus boreal&, bioassay of the red pigment-concentrating hormone. Marine Biol. 2, 13-18. FERNLUND P. and JOSEFSSON L. (1968) Chromactivating hormones of Pandalus borealis. Isolation and purification of the red pigment-concentrating hormone. Biochim. biophys. Acta 158, 262-273. FERNLUND P. and JOSEFSSON L. (1972) Crustacean
Locust corpora cardiaca peptides Color-change hormone: amino acid sequence and chemical synthesis. Science Wash. 177, 173-175. G~,DE G. and HOLW~RDA D. A. (1976) Involvement of adenosine Y,5"-cyclic monophosphate in lipid mobilization in Locusta migratoria. Insect Biochem. 6, 535--540. GOLDSWORTHYG. J., MORDUEW. and GUTHKELCHJ. (1972) Studies on insect adipokinetic hormones. Gen. comp. Endocr. 18, 545-551. HERMANW. S., CARLSENJ. B.,.CHRISTENSENM. and JOSEFSSON L. (1977) Evidence for an adipokinetic function of the RPCH activity present in the desert locust neuroendocrine system. Biol. Bull. mar. biol. lab., Woods Hole 153, 527-539. HOLWERDA D. A., VAN DOORN J. and BEENAKKERSA. M. T. (1977) Characterization of the adipokinetic and hyperglycaemic substances from the locust corpus cardiacum. Insect Biochem. 7, 151-157. JOSEESSONL., CARLSENJ., CHRISTENSENM. and HERMANW. S. (1977) Crustacean peptide hormones. Abstr. 1lth FEBS Meeting, Copenhagen 1977, Bl0-1, Ll.
501
MORDUE W. and GOLDSWORTHY G. J. (1969) The physiological effects of corpus cardiacum extracts in locust. Gen. comp. Endocr. 12, 360-369. MORDUE W. and STONE J. V. (1976) Comparison of the biological activities of an insect and a crustacean neurohormone that are structurally similar. Nature (Lond) 264, 287-289. MORDUE W. and STONE J. V. (1977) Relative potencies of locust adipokinetic hormone and prawn red pigmentconcentrating hormone in insect and crustacean systems. Gen. comp. Endocr. 33, 103-108. PENKE B., FERENCZ1 R. and KOVACS K. (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analyt. Bioehem. 60, 45-50. STONE J. V., MORDUE W., BATLEYK. E. and MORRIS H. R. (1976) Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature (Lond) 263, 207-211. THOMSEN M. (1946) Effect of corpus cardiacum and other insect organs on the colour-change of the shrimp, Leander adspersus. K. danske. Vidensk. Selsk. Skr. 19, 1-38.
0020-1700/79/0901-0497 $02.00/0
CHARACTERIZATION OF A SECOND PEPTIDE WITH ADIPOKINETIC AND RED PIGMENT-CONCENTRATING ACTIVITY FROM THE LOCUST CORPORA CARDIACA JENS CARLSEN,* WILLIAM S. HERMAN,t MOGENS CHRISTENSEN ++a n d EARS JOSEFSSON~ *Department of Biochemistry C, University of Copenhagen, Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark "tDepartment of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108, U.S.A. ++Novo Research Institute, Novo Alle, DK-2880 Bagsv~erd, Denmark (Received 4 December 1978) A new peptide with both adipokinetic activity in the locust and red pigment-concentrating activity in the shrimp, can be readily separated from the adipokinetic hormone in extracts of corpora cardiaca (CC) from Sehistoeerca americana gregaria by the use of gel chromatography and the Leander adspersus bioassay. The new peptide accounts for 20~, of the total biological activity in locust CC, and it is located principally in the CC glandular lobe. The amino acid composition of the pure peptide is: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. This composition is similar to, but not identical with the red pigmentconcentrating hormone of the shrimp. Key Word Index: Invertebrate peptide hormones, locust neurosecretory hormones, adipokinetic hormones in locust Abstract
INTRODUCTION
Hormone assays
LOCUST c o r p o r a cardiaca (CC) contain an adipokinetic h o r m o n e ( A K H ) that has been purified, characterized, a n d synthesized (STONE et al., 1976; BROOMFtELD and HARDY, 1977). Extracts from locust CC, as well as synthetic A K H also show hyperglycemic activity in cockroaches a n d red pigment-concentrating activity in crustaceans (MoRDUE and GOLDSWORTHY, 1969; MORDUE and STONE, 1976, 1977; THOMSEN, 1946). The possible existence in the locust C C of additional substances with the biological actions of synthetic A K H has been suggested on the basis of several experiments (MORDUE a n d GOLDSWORTHY, 1969; G,~DE a n d HOLWERDA, 1976), but attempts to separate a n d characterize such c o m p o u n d s have to date been unsuccessful (HOLWERDA et al., 1977). Using the highly sensitive, rapid, and convenient Leander adspersus e r y t h r o p h o r e bioassay (FERNLUND, 1968), we have t a k e n a d v a n t a g e of the red pigmentc o n c e n t r a t i n g activity o f A K H - l i k e substances (HERMAN et al., 1977) to isolate a n d characterize a second peptide with the biological effects of A K H from locust CC. O u r results are described below. MATERIALS AND METHODS
Adipokinetie response. Total haemolymph lipid was measured by the vanillin method (GOLDSWORTHV et al., 1972). Haemolymph (10 #1) was taken from a puncture in the membrane at the base of the hind leg. Sampling was followed by injection of a 50 #1 aliquot of the solution to be tested into the abdomen. After 1 hr a second 10 ~1 haemolymph sample was taken. Samples were immediately mixed with 0.4 ml concentrated sulphuric acid, and the mixtures were stirred thoroughly and analyzed for lipid content. Red pigment-concentrating activity. This was assayed on eyestalkless L. adsperus using a (2+2) point parallel line assay with ten animals at each dose (F~RNLUNO, 1968). Synthetic red pigment-concentrating hormone (RPCH) dissolved in 1.6~o (w/v) NaC1 was used as a standard. Concentrations of the standards were 0.47 × 10- ~o M and 1.41 × 10 ~0 M, respectively. Standards or samples (50 ill) to be tested were injected into each animal. A simplified procedure was used when assaying the separate tube fractions from column effluents and electrophoresis extracts for their activity. Samples of each fraction were appropriately diluted in 1.6",, (w/v) NaCI and 50/fl was injected into each of two animals at only one dose for each fraction. Activity responses were recorded as in the complete assay procedure. As no standard was included, no attempt was made to evaluate the quantitative amount of hormone in the different tube fractions of the column chromatographic separations. Thus, effluent curves show only the averaged responses for each tested fraction.
Experimental animals
Chemicals
Schistocerca americana gregaria were reared under crowded conditions with a 16 hr daily photo-period and a temperature of about 30°C. They were fed with bran, fresh grass and lettuce. Leander adspersus caught at the Danish coast were kept in l-m 3 closed system marine aquaria at 10°C. CC from freshly killed adult locusts were obtained by dissection. The locust Were decapitated and dissections carried out under chilled saline (0.128 M NaCI + 0.005 M KCI). The glands were stored at -20°C until extraction.
Sephadex G-25 fine and Sephadex LH-20 were purchased from Pharmacia, Uppsala, Sweden. Haemolymph lipid concentrations were determined using a Total Lipids kit obtained from Boehringer. All chemcials were of analytical grade. Water was delivered from a Milli-Q system, M illipore. Synthetic RPCH (FERNLUND and JOSEFSSON, 1972) was prepared in our laboratory (CHRISTENSEN, CARLSEN and JOSEFSSON, 1979) and had the same specific activity as the native hormone purified from L. adspersus (CARLSENel al., 1976). 497
JENS CARLSENet al.
498
Thin-layer chromatography Thin-layer chromatography (TLC) was performed on precoated TLC plates, Silica Gel 60 F-254 from E. Merck. Chromatograms were visualized by spraying with tert-butyl hypochlorite followed by p-tolidine/potassium iodide or with 10% (w/v) 4-N,N-dimethylamino-benzaldehyde in concentrated HCI, mixed with acetone (1:4, v/v) prior to use (Ehrlich reagent). The solvent systems used were; S-I; 2propanol-concentrated ammonia-water (8:1:1, by vol), S-2; ethyl aceta te-ethanol-water-pyridine-acetic acid (60:30:8:2:1, by vol), S-3; 2-propanol-water-acetic acid (25:10:1, by vol).
Amino acM analysis A Kontron Liquimat III, Kontron International Ziirich was used. Samples were hydrolized in sealed ampoules at I10°C for 24 hr in 6 M HCI. For determination of tryptophan, samples were hydrolyzed in 3 N mercaptoethane sulphonic acid (PENKEet al., 1974) and analyzed on a Beckmann Multichrom Liquid Column Chromatograph 4255.
Electrophoresis Glucose and alanine (serving as controls for the electrophoresis) were added and the samples applied on paper (Whatman No. 1). Electrophoresis was conducted at pH 2.3 [10% (v/v) acetic acid] and pH 6.0 (pyridine-acetic acid-water, 9:1:90, by vol) for 2 hr at 250 V. RESULTS
Preparation
of aqueous extract
CC were homogenized with ice-cold water (0.1 ml/CC) for 2 min in an all-glass Potter-Elvehjelm homogenizer, and the resulting suspension boiled for 5 min. After chilling on ice the suspension was centrifuged at 17,000 g for 15 min at 4°C. The supernatant was collected and the sediment extracted with a volume of water corresponding to 1/5 of the supernatant. The combined supernatants were frozen and thawed, and the small sediment formed removed by centrifugation. The resulting solution was lyophilized and 80 #g/CC of dry, pale yellow material obtained. The powder was extracted with water (0.4
ml/mg) at room temperature for 30 min. The insoluble material was centrifuged down (17,000 g, 15 min at 4°C) and the supernatant collected. The sediment was washed twice with a volume of water corresponding to 1/5 of the supernatant, before being discarded. The supernatants were combined: 1 ml of this aqueous extract corresponded to approx 25 CC.
Localization o f red pigment-concentrating activity and adipokinetic activity in gel filtration As shown in Fig. 1 red pigment-concentrating activity and adipokinetic activity are eluted over the same volume range from a G-25 column. The sensitive L. adspersus assay revealed that the red pigmentconcentrating activity could be separated; of the two distinct activities, peak I contained five times more activity than peak II. The same fractions also showed adipokinetic activity but two peaks could not be clearly demonstrated, probably on account of the reported limitations of the lipid mobilisation assay (HoLWERDA et al., 1977). Extracts prepared by ultrasonic treatment of the tissue in methanol according to SXONE et al. (1976) shows the same red pigment-concentrating activity 'pattern after gel filtration as the aqueous extract. In one experiment an aqueous extract prepared from Locusta migratoria C C was chromatographed on Sephadex G-25, and the same red pigmentconcentrating activity profile as described from S. americana gregaria CC was obtained. The localization of the red pigment-concentrating activity in separated glandular and storage lobes was investigated. Glandular lobes contain ten times more total activity than the corresponding storage lobes, but extracts from either part of the gland showed the same distribution between peak I and peak II as extracts from whole glands.
Preliminary characterization oJ the red pigmentconcentrating activities The aqueous extract (2 ml) with a red pigmentconcentrating activity corresponding to 2.0 #g R P C H
E
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' EFFLUENT(mll
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o
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Fig. 1 Gel filtration on Sephadex G-25 of the aqueous extract of corpora cardiaca. The extract (1 ml) was applied to an 1.4 cm i.d. x 36.0 cm column and eluted with water at 24°C. Fractions of 1.5 ml were collected and assayed. ( © - - © ) Red pigment-concentrating activity of the fractions assayed in L. adspersus at dilutions of 1:100. Adipokinetic activity assayed in S. americana gregaria with undiluted fractions. ( - - ) . Transmittance at 206 nm.
Locust corpora cardiaca peptides
499
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Fig. 2 Chromatography on LH-20 Sephadex. Column: 1.3 cm i.d. x 37.0 cm, eluted with 6'I. (v/vj lbutanol. A. Elution profile of compound I, fractions of 1.5 ml were collected, diluted 1:600and assayed for red pigment-concentrating activity. B. Elution profile of compound II, fractions of 1.5 ml were collected, diluted 1:100 and assayed for red pigment-concentrating activity.
was applied to a G-25 Sephadex column (2.5 cm i.d. x Purification oJ the active substances 92.5 cm) and eluted with water. Fractions of 8.4 ml Purification of the two substances with red pigmentwere collected. The two distinct red pigment- concentrating activity was achieved from 37 ml concentrating activities were completely separated; aqueous extract (corresponding to 920 CC) using the compound 1 eluted at 0.9 × V, (column volume) and procedure described in the preliminary charactericompound II, like RPCH (FERNLUNDand JOSEFSSON, zation. The column dimensions were: Sephadex G-25; 1968) retarded on the gel, eluted at 1.2 x Vt. The 5.0 cm i.d. × 89.0 cm and Sephadex LH-20; 2.5 cm i.d. active fractions in the two peaks were combined, and x 87.0 cm. Special care was taken to avoid evaporated to dryness under reduced pressure. The contamination from dust, etc., and the demineralized small residues were redissolved in 6~o (v/v) 1-butanol water was glass-distilled prior to use. After the final gel and subsequently chromatographed on a Sephadex filtration all solvent was evaporated and the residues LH-20 column eluted with 6% (v/v) 1-butanol. In Fig. taken in solution in a small volume of methanol 2 the elution profile of the two red pigment- (compond l: 1000 /A, compound II: 400 /A) and concentrating activities is shown. Compound I was analyzed. only slightly retarded on the gel and was eluted at 1.1 × Vt whereas compound II again behaved like RPCH Analysis of compound I and lI The methanolic solutions of the two isolated and (FERNLUND and JOSEFSSON, 1968) and was strongly retarded (elution vol: 1.8 × V,) in this purified compounds were examined by thin-layer chromatographic system. The combined active chromatography (reference: 1 #g synthetic RPCH) fractions (9 ml) after Sephadex LH-20 chromatog- and they were homogenous in all three media tested raphy of compound I, contained red pigment- (Table 1). The spots could also be developed spraying concentrating activity corresponding to 1.0 pg RPCH with Ehrlich reagent, indicating that the two peptides and 2 /~1 of the combined fractions increased the contain tryptophan. Amino acid analysis after acid haemolymph lipid concentration 14.8 _+ 0.9 mg/ml hydrolysis showed that compound I had the amino (mean _+ S.E.M., n = 6 ) when injected into S. acid composition: Asp2, Thr z, Glu, Pro, Gly, Leu, americana gregaria. The combined active fractions (13 Phe, and compound II the composition: Asp, Thr, Ser, ml) after Sephadex LH-20 chromatography of Glu, Gly, Leu, Phe (Table 2). Compound I (27 nmole) compound II contained red pigment-concentrating and compound II (25 nmole) (calculated from the amino activity corresponding to 0.2/~g RPCH and 10 pl of acid content) were obtained from 920 CC. Amino acid the combined fractions increased the haemolymph analysis, after hydrolysis in mercaptoethane sulphonic lipid concentration 8.1 + 1.5 mg/ml (mean _+ S.E.M., acid of a small amount (about 3 nmole) of the two peptides n = 6 ) when injected into S. americana gregaria. showed that tryptophan occurred in the same molar No difference was observed between compound I ratio as leucine. Quantitative assay of the purified and compound II on electrophoresis. When the papers peptides showed that they both are 1/50 as active as after electrophoresis at both pH values were cut in 0.5 cm RPCH when injected in L. adspersus, i.e. doses at pieces, extracted in 1.6% (w/v) NaCI and assayed for l x 10 -13 mole cause a significant response. Log red pigment-concentratingactivity, only extracts from dose-response curves were parallel with those pieces cut at the origin showed activity. obtained for RPCH.
500
JENS CARLSENet al.
Table 1. Rs values of compound I, compound I1 and RPCH. Approximately 1 #g each of the isolated hormones and the synthetic RPCH were spotted on each plate Solvent system S-1 S-2 S-3 Compound I Compound lI
0.37 0.42
0.14 0.34
0.63 0.69
RPCH
0.46
0.25
0.66
Table 2. Amino acid composition of the purified peptides. 100 pl of the methanolic solution of compound I and 200/~1of the methanolic solution of compound II were hydrolyzed Compound I nmole* Ratio Asp Thr Ser Glt~ Pro Gly Ala Cys Val Met lieu Leu Tyr Phe Lys His Arg
54.1 53.2 n.d. 26.1 25.1 31.4 n.d. n.d. n.d. n.d. n.d. 27.9 0.6 27.7 n.d. n.d. n.d.
Compound II nmole* Ratio
1.94 1.91 0.94 0.90 1.13 ----1.00 0.02 0.99
12.6 11.3 9.9 12.2 n.d. 12.9 n.d. n.d. n.d. 0.4 n.d. 12.2 n.d. 12.0 n.d. n.d. n.d.
1.03 0.93 0.81 1.00 -1.06 --0.03 -1.00 -0.98 ----
* n.d.: not detectable.
DISCUSSION Our demonstration that the locust corpus cardiacum contains two different biologically active peptides agrees with the early observation of MORDUE and GOLDSWORTHY (1969). Also chromatograms published by HOLWERDAet al. (1977), of extracts from Loeusta migratoria corpora cardiaca on Biogel P-2 show that the adipokinetic activity is eluted over a wide volume range, indicating lack of homogeneity. O f the two adipokinetic active peptides isolated, compound I has an amino acid composition identical with the composition of the A K H purified from S. americana gregaria and L. migratoria CC glandular lobes by STONE et al. (1976). This hormone is a decapeptide with the following structure: pGlu-LeuAsn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH 2 confirmed by chemical synthesis (BROOMFIELD and HARDY, 1977). C o m p o u n d II is probably an octapeptide with the following amino acids in equimolar amounts: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. The tryptophan content is demonstrated by the positive reaction with Ehrlich reagent on the T L C plates, the presence of tryptophan in amino acid analysis after hydrolysis in 2-mercaptoethane sulphonic acid, and by the red
pigment-concentrating activity since tryptophan is indispensible for the red pigment-concentrating activity (JOSEFSSON et al., 1977, CHRISTENSEN et al., 1978). The composition of compound Il is almost identical with RPCH: pGlu-Leu-Asn-Phe-Ser-ProGIy-Trp-NH 2 (FERNLUND and JOSEFSSON, 1972), and shows the same characteristic behaviour when chromatographed on Sephadex gels, indicating a structure very similar to RPCH. C o m p o u n d II like A K H and RPCH, is electrophoretically immobile at neutral and acid pH. These data prove that, like A K H and RPCH, compound II has blocked NH2-terminus and no free carboxyl groups, i.e. a blocked C O O H terminus and the aspartic acid residue as its amide. C o m p o u n d II in the present study has the same red pigment-concentrating activity as A K H (compound I) in L. adspersus. C o m p o u n d II also has, estimated from the relation between the lipid mobilization effect and the red pigment-concentrating activity, about the same adipokinetic activity as A K H in S. americana gregaria. The amount of compound II/CC, calculated from the distribution in the Sephadex G-25 gel filtration, is 1/5 of A K H , but the yield in the purification is lower. C o m p o u n d 11 is located in both the glandular and the storage lobes but occurs as A K H mainly in the glandular lobe. Owing to the minimal amount of material available (about 0.03 #g pure peptide was obtained from one S. americana gregaria), it was not possible in this study to elucidate the structure of compound II. But using the purification system described, A K H is easily separated, so purification of amounts allowing for structural analysis should be possible. This study also opens up for investigation the possible physiological role of this second peptide with adipokinetic activity in the locust neuroendocrine system. Acknowledgement~We thank Ms B. HARDERfor skillful technical assistance, and E. LUNDfor the amino acid analysis. The investigation was aided by grants from the Danish Natural Science Research Council (Project No. 3565) and the University of Minnesota Graduate School. USPHS grant HD-07336. REFERENCES BROOMFIELDC. E. and HARDYP. M. (1977) The synthesis of locust adipokinetic hormone. Tetrahedron Lett. 25, 2201-2204. CARLSEN J., CHR1STENSEN M. and JOSEFSSON L. (1976) Purification and chemical structure of the red pigmentconcentrating hormone of the prawn Leander ad6lwrsus. Gen. comp. Endocr. 30, 327-331. CHRISTENSEN M., CARLSEN J. and JOSEFSSON L. (1978) Structure-function studies on red pigment-concentrating hormone; The significance of the terminal rc~Jdues. HoppeSeyler's Z. physiol. (;hem. 359, 813-818. CHRISTENSEN M., CARLSENJ. and JOSEFSSONL, Structurefunction studies on red pigment-concentrating hormone. I1. The significance of the C-terminal tryptophan amide. Hoppe-Seyler's Z. physiol. Chem. (In press). FERNLUNDP. (1968) Chromactivating hormones of Pandalus boreal&, bioassay of the red pigment-concentrating hormone. Marine Biol. 2, 13-18. FERNLUND P. and JOSEFSSON L. (1968) Chromactivating hormones of Pandalus borealis. Isolation and purification of the red pigment-concentrating hormone. Biochim. biophys. Acta 158, 262-273. FERNLUND P. and JOSEFSSON L. (1972) Crustacean
Locust corpora cardiaca peptides Color-change hormone: amino acid sequence and chemical synthesis. Science Wash. 177, 173-175. G~,DE G. and HOLW~RDA D. A. (1976) Involvement of adenosine Y,5"-cyclic monophosphate in lipid mobilization in Locusta migratoria. Insect Biochem. 6, 535--540. GOLDSWORTHYG. J., MORDUEW. and GUTHKELCHJ. (1972) Studies on insect adipokinetic hormones. Gen. comp. Endocr. 18, 545-551. HERMANW. S., CARLSENJ. B.,.CHRISTENSENM. and JOSEFSSON L. (1977) Evidence for an adipokinetic function of the RPCH activity present in the desert locust neuroendocrine system. Biol. Bull. mar. biol. lab., Woods Hole 153, 527-539. HOLWERDA D. A., VAN DOORN J. and BEENAKKERSA. M. T. (1977) Characterization of the adipokinetic and hyperglycaemic substances from the locust corpus cardiacum. Insect Biochem. 7, 151-157. JOSEESSONL., CARLSENJ., CHRISTENSENM. and HERMANW. S. (1977) Crustacean peptide hormones. Abstr. 1lth FEBS Meeting, Copenhagen 1977, Bl0-1, Ll.
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MORDUE W. and GOLDSWORTHY G. J. (1969) The physiological effects of corpus cardiacum extracts in locust. Gen. comp. Endocr. 12, 360-369. MORDUE W. and STONE J. V. (1976) Comparison of the biological activities of an insect and a crustacean neurohormone that are structurally similar. Nature (Lond) 264, 287-289. MORDUE W. and STONE J. V. (1977) Relative potencies of locust adipokinetic hormone and prawn red pigmentconcentrating hormone in insect and crustacean systems. Gen. comp. Endocr. 33, 103-108. PENKE B., FERENCZ1 R. and KOVACS K. (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analyt. Bioehem. 60, 45-50. STONE J. V., MORDUE W., BATLEYK. E. and MORRIS H. R. (1976) Structure of locust adipokinetic hormone, a neurohormone that regulates lipid utilisation during flight. Nature (Lond) 263, 207-211. THOMSEN M. (1946) Effect of corpus cardiacum and other insect organs on the colour-change of the shrimp, Leander adspersus. K. danske. Vidensk. Selsk. Skr. 19, 1-38.