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Changes and characteristics of the crustacean hyperglycemic hormone (CHH material) in Palaemon serratus pennant (crustacea, decapoda, natantia) during the different steps of the purification
Comp. Biochem. Physiol. Vol. 79B, No. 3, pp. 353-360, 1984 Printed in Great Britain
0305-0491/84$3.00+ 0.00 Pergamon Press Ltd
CHANGES AND CHARACTERISTICS OF THE CRUSTACEAN HYPERGLYCEMIC HORMONE (CHH MATERIAL) IN P A L A E M O N SERRATUS PENNANT (CRUSTACEA, DECAPODA, NATANTIA) DURING THE DIFFERENT STEPS OF THE PURIFICATION A. VAN WORMHOUDT,* F. VAN HERP,J" C. BELLON-HUMBERT* and R. KELLER3~ *Laboratoire de Biologie Marine, Collrge de France, 29110 Concarneau, France (Tel: 98-97-1232); tZoologisch Laboratorium, Kath. Universiteit, Toernooiveld, 6525 ED Nijmegen, Nederland; and :~Institut fur Zoophysiologie, Universitiit Bonn, D-5300 Bonn 1, Bundesrepublik Deutschland (Received 8 March 1984)
Abstract--1. The importance of the MTGX2 sinus gland complex in the production and the storage of CHH material has been demonstrated by bioassays using extracts of the different eyestalks structure. 2. The purification of the hyperglycemicmaterial (CHH) was carried out in Palaemon serratus by two procedures, starting from total eyestalk extracts or from extirpated sinus glands. 3. There is evidence for the presence of 3 different forms of the hyperglycemicactive fractions expressed by their electrophoretic mobility and molecular weight. 4. The main fraction has a molecular weight of around 8000 and a rm of 0.59 in Davis electrophoresis but small peptides (around 2000 mol. wt) are also hyperglycemicif injected in eyestalkless shrimps.
INTRODUCTION
After the red pigment concentrating hormone (Fernfund and Josefsson, 1972; Fernlund, 1974) and the light adaptating distal retinal pigment hormone (Fernlund, 1976), the hyperglycemic hormone (CHH) became the third biochemically best-known hormone in Crustacea (Kleinholz and Keller, 1973). Attempts to isolate this neurohormone have resulted in reasonably pure preparations (Kleinholz and Keller, 1973; Skorkoswki et al., 1977; Keller and Wunderer, 1978) for several crustacean species. Analysis of the amino acid composition of the main form of hyperglycemic factor was carried out for Cancer (Kleinholz, 1975), Carcinus and Orconectes (Keller and Wunderer, 1978; Keller and Sedlmeier, 1978; Keller, 1981). Moreover, a molecular heterogeneity for CHH in one species was already reported for Cancer by Kleinholz and Keller (1978), Pascifastacus and Orconectes by Keller (1976, 1977) and for Crangon by Skorkowski et al. (1977). With regard to the molecular weight, Keller 098 l) found a comparable mol. wt of about 6800 for Carcinus and Orconectes, Skorkowski et al. (1977) estimated two molecular forms with an average respectively of 20,500 and 7300 mol. wt in Crangon. Trausch and Bauchau (1981) isolated a small peptide from Homarus, which had a hyperglycemic activity and a molecular weight of 1500. Preliminary results on Palaemon serratus gave indications for the polymorphism of this hormone (Van Wormhoudt et al., 1978, 1981). Already in 1944, Abramowitz et al. had pointed out that the main source of the crustacean hyperglycemic hormone was the X organ-sinus gland complex of the eyestalk. The precise localization of the CHH was possible in the last years by immunocytochemical procedures (Van Herp and Van Buggenum, 1979; Jaros and Keller, 1979; Gorgels-Kallen and Van Herp, 1981; Gorgels-Kallen et al., 1982).
In this paper, the ultimate aim was to study the hyperglycemic material in the eyestalk of the prawn Palaemon serratus, using two procedures of purification, already reported by Kleinholz and Keller (1973) from total eyestalk extracts and by Keller and Wunderer (1978), and Keller (1981) from extirpated sinus gland. MATERIALS AND METHODS
Extraction and purification from the total eyestalk For the extraction and preliminary fractionation of 25 g of lyophilized Palaemon eyestalks, corresponding to 8500 eyestalks, the procedure of Kleinholz and Keller (1973) was used with a slight modification. After ammonium sulphate precipitation, the precipitate was suspended in ice-cold Tris-HC1 buffer (0.05 M, pH 8.0) containing 0.1% (v/v) 2,2' thiodiethanol, and dialyzed against the same buffer until the solution was free of sulphate ions (Thomas dialysing tube, mol. wt cut-off: 3500). The acid precipitation step at pH 4.5 with diluted acetic acid as described by Kleinholz and Keller, was omitted. A preparative polyacrylamide gel electrophoresis was carried out according to Davis (1964) in a 6 mm thick slab gel (separation gel of 7% and spacer gel of 4%). After electrophoresis at 4°C, the gel was cut horizontally into slices of 10mm. These segments were reduced to small pieces and extracted in distilled water at 4°C for 24 hr. The eluates were separated from the gel pieces by filtration and lyophilized. The obtained fractions corresponding to 2 equivalent-eyestalkswere tested for their biological activity on the blood glucose content. The CHH containing fractions were combined and the quantity of protein determined by the method of Lowry et al. (1951), using bovine serum albumin as calibration standard. Extraction and purification of CHH from extirpated sinus glands From fresh Palaemon eyestalks, 1237 sinus glands were dissected, collected in distilled water at 0°C in a ground glass micro tissue grinder (Kontes) and lyophilized. For the extraction, the procedure of Keller and Wunderer (1978)
353
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was used without modifications. The obtained lyophilized material was dissolved in 100 mM ammonium acetate buffer (pH8.5) and desalted on a Sephadex G-10 column (0.9 x 13.5 cm) equilibrated with the same buffer. For preparative polyacrylamide electrophoresis of that material a disc-electrophoresis was carried out using 7~o gels in 3 tubes (0.7 x 8 cm). The material was incorporated into sample gels which were photopolymerized. Electrophoresis (3 mA/gel) was stopped after a migration of 65 mm was obtained and 45 gel slices of 1.5 mm were cut out in each gel. They were twice eluted in 1 ml of extraction buffer. The supernatants were lyophilized and the frozen dried material was solubilized in distilled water to test the hyperglycemic activity. Approximately one sinus gland equivalent was injected into each test animal.
Gel chromatograph)' To compare the molecular weight of several hyperglycemic active fractions, gel chromatography on Sephadex G-50 (Superfine) was performed. At first, the column (0.6 x 140cm) was equilibrated with 10mM ammonium acetate (pH 8.5) and calibrated with RNase (13,000), insulin (5700), glucagon (3550), bacitracin (1411). The flow rate was 2.2 ml/hr and the fraction volume was 1 ml. The absorbance of each fraction was read at 230nm with a Zeiss spectrophotometer.
Animals and starting material Prawns were collected in the bay of Concarneau and kept in running sea water in the laboratory. They were selected according to the intermolt C stage and to the size (about 6 cm). To test the hyperglycemic activity, unfed and eyestalkless animals, from which eyestalks were cut off 18 hr before injections, were used. All the test fractions were injected (10#l/animal) into the abdominal muscles. The haemolymph (about 50 #l/animal) was collected 2 hr after the injection by section of the telson and uropods. After deproteinization of the blood samples with 0.5 M perchloric acid, the glucose concentration was estimated by the Godperid test (Boehringer, Mannheim). To study the overall distribution of the hyperglycemic active material in the eyestalk, different structures (medulla terminalis, medulla interna, medulla externa, MTGX-1 organ region, MTGX-2 organ region, medulla externa X organ, organ of Bellonci and sinus gland) were dissected and collected in distilled water in a micropotter. After homogenization and centrifugation in a Beckman Microfuge at 10,000rpm, the supernatants were recovered. Hyperglycemic activity was tested after injection of 1 organ equivalent for each structure. The protein content per structure was measured by the method of Lowry et al. (t951).
RESULTS
Localization of the hyperglycemic activity in the structures of the eyestalk As shown in Table 1, selective injections of eyestalk structures indicate a hyperglycemic activity in the extracts of the total eyestalk, total medulla terminalis (MT) and externa (ME), respectively in the regions of the medulla ganglionic X organs 1 (MTGX-1) and 2 ( M T G X - 2 ) and the neurohaemal sinus gland (SG). On the contrary, injections of extracts of the medulla externa X organ (MEX) and of the organ of Bellonci (OB) remain comparable to injections of sea water (controls).
Purification of the hyperglycemic factor (s) From eyestalks. The detection of C H H activity
Table 1. Localization of CHH activity in the eyestalk structures ol Palaemon
Control Total eyestalks Medulla terminalis Medulla interna Medulla externa MTGX-I MTGX-2 MEX OB SG
serrattgs
Protein per structure (l~g)
]Lg glucose ml haemolymph +_ SEM)
980 76 10.5 31 21 68 5.3 4.4 8.4
37 * 26(7) 235 ± 93 17) 190 * 75 (51 54 + 15 121 2411 ~ 40 (2) 22 + 5 (2) 167 k 45 (2) 5~~ + 17t31 40 ± 30(4~ 215 +__82 (5)
The extracts were prepared in a micropotter with distilled water and centrifuged in Beckman microfuge at 10,000 rpm. Between brackets: number of experiments in which animals were injected with 1 organ equivalent. MTGX-I, MTGX-2: neurosecretory cell-groups 1 and 2 of the medulla terminalis called medulla terminalis ganglionaris X organs. MEX: Neurosecretory cell group of the medulla externa called medulla externa-X organ. OB: organ of Bellonci; SG: sinus gland.
after the purification procedure of Kleinholz and Keller (1973) was performed by bioassays for hyperglycemia in the fractions of the preparative electrophoresis step. As shown in Fig. la, a very high hyperglycemic response was induced after the injections of gel fractions 7 and 8, corresponding to 2 eyestalk equivalents each. These hyperglycemic fractions show a r~ index between 0.55 and 0.60 determined on the electrophoretic migration of these 2 fractions in a polyacrylamide gel (Fig. lb, c). A lower hyperglycemic activity can be attributed to an electrophoretical fraction of a rm index 0.30 and to a r,, index 0.71. By combining the 2 hyperglycemic fractions (7 and 8) a total protein content of 8.5 mg protein was found but was probably largely overestimated due to the Folin reagent. The molecular weights of these 3 fractions were determined immediately using gel filtration on Sephadex G 50 (superfine). In the three cases, the main hyperglycemic activity eluted with a elution volume of between 26 and 28 ml thus corresponding to a molecular weight of 8000. From extirpated sinus glands. Following the first steps of the procedure of Keller and Wunderer (1978) and Keller (1981), consisting of desaltation on Sephadex G 10 and a preparative polyacrylamide gel electrophoresis from sinus gland material, lyophilized and only extracted immediately before electrophoresis, the main hyperglycemic activity can be related to a protein band with a r,, index between 0.58 and 0.60 (Fig. 2 a, b). A lower hyperglycemic activity was detected in the gel region corresponding to protein material with a r m value of 0.71. However, after carrying out comparable electrophoresis from the same extract of sinus glands, after its storage for a long period at - 20°C, a hyperglycemic activity was detected only in an electrophoretic fraction with an index of 0.71 (Fig. 3a, b). The hyperglycemia produced by this fraction was twice as high as the reaction provoked by the biologically active fraction with an rm index 0.58-0.60. When the fractions containing C H H were pooled the protein content determined was found to be 234 ~g.
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Molecular weights were estimated for the C H H active fraction with a r,, index 0.71, using gel filtration on Sephadex G 50 (Superfine) (Fig. 4) and compared with the elution pattern of freshly prepared sinus gland extracts (Fig. 5). F o r the fraction 0.71, the apparent molecular weight oscillates between 2000
and 3000, taking into account the highest hyperglycemic activity eluted at a volume of 42 ml. Moreover, a smaller hyperglycemic fraction around the void volume of the column was determined. In the same system the red pigment concentrating hormone (RPCH) from total sinus gland extract eluted in a
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Dose-response relations of the hyperglycemic factors To compare the hyperglycemic activity of the two fractions described (rm 0.59 prepared by eyestalks extraction and rm 0.71 prepared by sinus gland extraction), these fractions were injected at different concentrations. In Table 2 a bioassay shows that the induced hyperglycemic responses are proportional to the calculated amount of injected hormone.
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DISCUSSION
The presence of the hyperglycemic activity in the eyestalk structures revealed by bioassays confirms our preliminary histo-immunological results in Palaemon serratus, using an antiserum against CHH from the crayfish Astacus leptodactylus (Van Wormhoudt et al., 1981). These results are also in agreement with the comparative immunocytochemical investigations of the crustacean hyperglycemic hormone of some decapod Crustacea by GorgelsKallen et al. (1982). An immunocytochemical reaction is present in a considerable portion of the sinus gland in the MTGX-2sinus gland nerve and in a number of neurosecretory cells in the MTGX-2. In this way, the induced hyperglycemic activity by the extracts from the MTGX-2 region and that from the sinus gland were expected, while the reactivity in the total extract of the medulla terminalis, as well as in the extract of the MTGX-1 region, is probably related to the pathway of the X organ-sinus gland tract containing CHH. The hyperglycemia provoked by the total extract of the medulla externa cannot be explained for the moment, since no positive cells for the anti-CHH serum were observed in the MEX neurosecretory cells of Palaemon serratus. Moreover,
this organ does not induce hyperglycemia (BellonHumbert et al., 1981). Purification of hyperglycemic material according to Kleinholz and Keller (1973) using total eyestalk extracts and according to Keller and Wunderer (1978) and to Keller (1981) using sinus gland material gives evidence for 3 forms of CHH. Yet, a major neuropeptide hormone with CHH activity and a rm index 0.59 in the polyacrylamide gel electrophoresis (7.5~) (Davis, 1964) is determined. This index represents an intermediary electrophoretic mobility between the indices of the Astacidae (r,, 0.35) and the Brachyura (r m 0.69) stated by Keller (1977). For the prawn Pandalus, a comparable r,, value 0.62 can be calculated from the results of Kleinholz and Keller (1973). The molecular weight estimation of this hyperglycemic fraction in the freshly prepared sinus gland material and after eyestalk purification allows us to determine its molecular weight at around 8000. Comparing these results on the hyperglycemic material from Palaemon serratus with related data in literature, we can deduce some homology with the results of Kleinholz and Keller (1973) who found an average of 6300 mol. wt for Pandalus, Skorkowski et al. (1977) with 7300 mol. wt in Crangon, Keller and Wunderer (1978) with 6726 mol. wt for Carcinus and
Crustacean hyperglycemic hormone
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Keller (1981) with 6812 mol. wt for Orconectes. However, after freezing and storage of the sinus gland extracts, the main highest hyperglycemic activity was correlated with an electrophoretical fraction with arm 0.71 and a molecular weight of 2000-3000. Some authors also reported hyperglycemic activity of small peptides at least in some conditions (Mordue and Stone, 1977; Carlsen et al., 1977; Loughton and Orchard, 1981). In Homarus, Trausch and Bauchau (1981) described small molecular peptides from eyestalks of about 1500 mol. wt with a hyperglycemic activity. This was obtained also after long storage and freezing and in the two cases they could correspond to partial hydrolysis of the main form of the hyperglycemic factor of 5000-7000 mol. wt recovered later in Homarus sinus glands (Van Deynen et al., 1983). Whether all these peptides are physiological can be studied in relation to prohormone and release studies in crustacean. In this way, the existence in the eyestalk of high molecular weight peptides referred by Skorkowki et al. (1977) and Andrew and Saleuddin (1979) should be reconsidered and also the existence of a polymorphism of the hormone as described
recently on reverse HPLC (Newcomb, 1983; Stuenkel, 1983). Comparing the 2 used procedures for purification, from the total eyestalk and from the sinus gland extracts, in relation to the protein recovery for CHH material, measured by the method of Lowry (1951), we find about 1 #g Lowry per eyestalk and 0.2/lg Lowry per sinus gland (between 0.5 and 1.3/lg according to different experiments). These results are
Table 2. Hyperglycemic effects of different concentrations of C H H extracts in Palaemon serratus ng protein (Lowry) injected
Eyestalk CHH r m 0.59
Sinus gland CHH rm 0.71
Control 50 500 5000
25 q: 9 38* 98* 230 :t: 30
62 q: 10 105 q: 20 170 q: 30 220 T 20
Blood glucose is expressed in #g/ml of haemolymph and corresponds to a pool of 2 x 5 prawns,* expected. Protein estimated by Lowry, largely overestimated C H H content (Keller, 1981).
360
A. VAN WORMHOUDT e l al.
in agreement with the d a t a from Skorkowski et al. (1977) for Crangon, w h o f o u n d a b o u t 1.2/~g Lowry per eyestalk a n d with the results of Keller and W u n d e r e r (1978) for Carcinus with 0.26 # g per sinus gland. The differences in recovery between the 2 procedures must be related to the p r o p e r characteristics of each purification step. F o r example, the recovery o f material from polyacrylamide gels is not always easy while dialysis of the eyestalk material was carried o u t with dialysis m e m b r a n e s with a cut off of 3500 mol. wt. Meanwhile, by R.I.A., Jaros a n d Keller (1979) have f o u n d approximately 1.2 # g of C H H in Carcinus sinus gland, using an a n t i s e r u m against Carcinus C H H . This a m o u n t is c o m p a r a b l e to the a m o u n t of 1 p g we have recovered per total eyestalk by the modified procedure of Kleinholz a n d Keller (1973), but is higher t h a n the value given for O r conectes (Keller, 1981) a r o u n d 0 . 0 3 # g Lowry per sinus gland. Fifty n a n o g r a m m e s of C H H (r,, 0.59 or r m 0.71) m e a s u r e d by Lowry are effective to stimulate at least twice the glycemia c o n t e n t in p r a w n s of 2 g b u t the exact value of pure C H H is not known. Lower values are o b t a i n e d for Carcinus (Keller a n d W u n d e r e r , 1977) or for Uca (Keller, 1981). This difference could be due to the instability of the molecule before and d u r i n g the injection or some seasonal influence (Leuven et al., 1982). REFERENCES
Abramowitz A. A., Hisaw F. L. and Papandrea D. N. (1944) The occurrence of a diabetogenic factor in the eyestalk of crustaceans. Biol. Bull. 86, 1-5. Andrew R. D. and Saleuddin A. S. M. (1979) Two dimensional gel electrophoresis of neurosecretory potypeptides in crustacean eyestalk. J. Comp. Physiol. 134, 303 313. Bellon-Humbert C., Van Herp F., Strolenberg G. E. C. M. and Denuce J. M. (1981) Histological and physiological aspects of the neurosecretory cell-group MEX (medulla externa X organ) in the eyestalk of Palaemon serratus (Crustacea: Decapoda: Natantia). Biol. Bull. 1611, 11-30. Carlsen J., Herman W. S., Christensen M. and Josefsson L. (1979) Characterization of a second peptide with adipokinetic and red pigment concentration activity from the locust Corpora eardiaca. Insect Biochem. 9, 497-501. Davis B. J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sei. 121, 404-427. Fernlund P. (1974) Structure of the red pigment concentrating hormone of the shrimp, Pandalus borealis. Bioehem. biophys. Acta 371, 304-311. Fernlund P. (1976) Structure of a light adaptating hormone from the shrimp Pandalus borealis. Biochim. biophys. Acta 439, 17-25. Fernlund P. and Josefsson L. (1972) Crustacean color change hormone: amino acid sequence and chemical synthesis. Science 177, 173-175. Gorgels-Kallen J. L., Van Herp F. and Leuven R. (1982) A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod Crustacea. J. Morph. 174, 161-168. Jaros P. and Keller R. (1979) Immunocytochemical identification of hyperglycemic hormone producing cells in the eyestalk of Carcinus maenas. Cell Tissue Res. 204, 379-385. Keller R. (1976) Electrophoretic analysis of neurosecretory substances 1¥om the sinus gland of decapod crustaceans. Colloq. Intern. CNRS. Lille 251, 247-254.
Keller R. (1977) Comparative etectrophoresis studies of crustacean neurosecretory hyperglycemic and melanophore stimulating hormones from isolated sinus glands..l. comp. Physiol. 122, 359-373. 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. 141, 445 450. Keller R. and Sedlmeier D. (1978) Hyperglycemic hormones in crustaceans. In Comparative Endocrinology (Edited by Gaillard P. J. and Boer H. H.), pp. 437 440. Elsevier/North-Holland, Amsterdam. Keller R. and Wunderer G. (1978) Purification and amino acid composition of the neurosecretory hyperglycemic hormone from the sinus gland of the shore crab Carcinus maenas. Gen. comp. Endocr. 34, 32 335. Kleinholz L. H. (1975) Purified hormones from the crustacean eyestalk and their physiological specificity. Nature. Lond. 258, 256-257. Kleinholz L. H. and Keller R. (1973) Comparative studies in crustacean neurosecretory hyperglycemic hormones. Gen. comp. Endocr. 21, 554-564. Leuven R. S. E. W., Jaros P. P., Van-Herp F. and Keller R. (1982) Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. Gen. comp. Endocr. 46, 288-296. Loughton B. G. and Orchard I. (1981) The nature of the hyperglycemic factor from the glandular lobe of the corpus cardiacum of Locusta migratoria. J. lnsect. Physiol. 27, 383 385. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. Mordue W. and Stone J. V. (1977) Relative potencies of locust adipokinetic hormone and prawn red pigment concentrating hormone in insect and crustacean system. Gen. comp. Endocr. 33, 103-108. Newcomb R. W. (1983) Peptides in the sinus gland of Cardisoma carnifex. Isolation and amino acid analysis. J. comp. Physiol. 153, 207-221. Skorkowski E. F., Rykiert M. and Lipinska B. (1977) Hyperglycemic hormone from the eyestalk of the shrimp Crangon crangon. Gen. comp. Endocr. 33, 460-466. Stuenkel E. L. (1983) Biosynthesis and axonal transport of protein in the X organ sinus gland complex. J. comp. Physiol. 153, 191-205. Trausch G. and Bauchau R. (1981) Biological activity of eyestatk extracts the lobster Homarus americanus. In X l Conference E.S.C.E. Jerusalem, August 1981. Van Deynen J. F., D. Soyez and F. Van-Herp. (1983) Partial purification of 4 crustacean neurohormones (MIH, GIH. CHH and RCPH) by HPLC. Colloque intern. CNRS. Biosynthesis, metabolism action of invertebrate hormones. Strasbourg August-September 2/9/1983. Van Herp F. and Van Buggenum H. J. M. (1979) Immunocytochemical localization of the hyperglycemic hormone (HGH) in the neurosecretory system of the eyestalk of the crayfish Astacus leptodactylus. Experientia 35, 1527-1528. Van Wormhoudt A., Van Herp F., Bellon C., Keller R. and Jaros P. (1981) Purification, caract6risation et localisation de l'hormone hyperglyc+mique chez Palaemon serratus. VII r6union carcinologistes, Banyuls S/Mer, 1-6 Juin 1981. Van Wormhoudt A., Van Herp F., Bellon C.. Strolenberg G. E. C. M. and Venema D. (1978) Cyclic variations of the glucose concentration in the hemolymph and studies on the hyperglycemic hormone of Palaemon serratus. In Comparative Endocrinology (Edited by Gaillard P. H. and Boer H. H.) Eighth International Symposium Comparative Endocrinology, Amsterdam, June 19-23, p. 174. 1978.
0305-0491/84$3.00+ 0.00 Pergamon Press Ltd
CHANGES AND CHARACTERISTICS OF THE CRUSTACEAN HYPERGLYCEMIC HORMONE (CHH MATERIAL) IN P A L A E M O N SERRATUS PENNANT (CRUSTACEA, DECAPODA, NATANTIA) DURING THE DIFFERENT STEPS OF THE PURIFICATION A. VAN WORMHOUDT,* F. VAN HERP,J" C. BELLON-HUMBERT* and R. KELLER3~ *Laboratoire de Biologie Marine, Collrge de France, 29110 Concarneau, France (Tel: 98-97-1232); tZoologisch Laboratorium, Kath. Universiteit, Toernooiveld, 6525 ED Nijmegen, Nederland; and :~Institut fur Zoophysiologie, Universitiit Bonn, D-5300 Bonn 1, Bundesrepublik Deutschland (Received 8 March 1984)
Abstract--1. The importance of the MTGX2 sinus gland complex in the production and the storage of CHH material has been demonstrated by bioassays using extracts of the different eyestalks structure. 2. The purification of the hyperglycemicmaterial (CHH) was carried out in Palaemon serratus by two procedures, starting from total eyestalk extracts or from extirpated sinus glands. 3. There is evidence for the presence of 3 different forms of the hyperglycemicactive fractions expressed by their electrophoretic mobility and molecular weight. 4. The main fraction has a molecular weight of around 8000 and a rm of 0.59 in Davis electrophoresis but small peptides (around 2000 mol. wt) are also hyperglycemicif injected in eyestalkless shrimps.
INTRODUCTION
After the red pigment concentrating hormone (Fernfund and Josefsson, 1972; Fernlund, 1974) and the light adaptating distal retinal pigment hormone (Fernlund, 1976), the hyperglycemic hormone (CHH) became the third biochemically best-known hormone in Crustacea (Kleinholz and Keller, 1973). Attempts to isolate this neurohormone have resulted in reasonably pure preparations (Kleinholz and Keller, 1973; Skorkoswki et al., 1977; Keller and Wunderer, 1978) for several crustacean species. Analysis of the amino acid composition of the main form of hyperglycemic factor was carried out for Cancer (Kleinholz, 1975), Carcinus and Orconectes (Keller and Wunderer, 1978; Keller and Sedlmeier, 1978; Keller, 1981). Moreover, a molecular heterogeneity for CHH in one species was already reported for Cancer by Kleinholz and Keller (1978), Pascifastacus and Orconectes by Keller (1976, 1977) and for Crangon by Skorkowski et al. (1977). With regard to the molecular weight, Keller 098 l) found a comparable mol. wt of about 6800 for Carcinus and Orconectes, Skorkowski et al. (1977) estimated two molecular forms with an average respectively of 20,500 and 7300 mol. wt in Crangon. Trausch and Bauchau (1981) isolated a small peptide from Homarus, which had a hyperglycemic activity and a molecular weight of 1500. Preliminary results on Palaemon serratus gave indications for the polymorphism of this hormone (Van Wormhoudt et al., 1978, 1981). Already in 1944, Abramowitz et al. had pointed out that the main source of the crustacean hyperglycemic hormone was the X organ-sinus gland complex of the eyestalk. The precise localization of the CHH was possible in the last years by immunocytochemical procedures (Van Herp and Van Buggenum, 1979; Jaros and Keller, 1979; Gorgels-Kallen and Van Herp, 1981; Gorgels-Kallen et al., 1982).
In this paper, the ultimate aim was to study the hyperglycemic material in the eyestalk of the prawn Palaemon serratus, using two procedures of purification, already reported by Kleinholz and Keller (1973) from total eyestalk extracts and by Keller and Wunderer (1978), and Keller (1981) from extirpated sinus gland. MATERIALS AND METHODS
Extraction and purification from the total eyestalk For the extraction and preliminary fractionation of 25 g of lyophilized Palaemon eyestalks, corresponding to 8500 eyestalks, the procedure of Kleinholz and Keller (1973) was used with a slight modification. After ammonium sulphate precipitation, the precipitate was suspended in ice-cold Tris-HC1 buffer (0.05 M, pH 8.0) containing 0.1% (v/v) 2,2' thiodiethanol, and dialyzed against the same buffer until the solution was free of sulphate ions (Thomas dialysing tube, mol. wt cut-off: 3500). The acid precipitation step at pH 4.5 with diluted acetic acid as described by Kleinholz and Keller, was omitted. A preparative polyacrylamide gel electrophoresis was carried out according to Davis (1964) in a 6 mm thick slab gel (separation gel of 7% and spacer gel of 4%). After electrophoresis at 4°C, the gel was cut horizontally into slices of 10mm. These segments were reduced to small pieces and extracted in distilled water at 4°C for 24 hr. The eluates were separated from the gel pieces by filtration and lyophilized. The obtained fractions corresponding to 2 equivalent-eyestalkswere tested for their biological activity on the blood glucose content. The CHH containing fractions were combined and the quantity of protein determined by the method of Lowry et al. (1951), using bovine serum albumin as calibration standard. Extraction and purification of CHH from extirpated sinus glands From fresh Palaemon eyestalks, 1237 sinus glands were dissected, collected in distilled water at 0°C in a ground glass micro tissue grinder (Kontes) and lyophilized. For the extraction, the procedure of Keller and Wunderer (1978)
353
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was used without modifications. The obtained lyophilized material was dissolved in 100 mM ammonium acetate buffer (pH8.5) and desalted on a Sephadex G-10 column (0.9 x 13.5 cm) equilibrated with the same buffer. For preparative polyacrylamide electrophoresis of that material a disc-electrophoresis was carried out using 7~o gels in 3 tubes (0.7 x 8 cm). The material was incorporated into sample gels which were photopolymerized. Electrophoresis (3 mA/gel) was stopped after a migration of 65 mm was obtained and 45 gel slices of 1.5 mm were cut out in each gel. They were twice eluted in 1 ml of extraction buffer. The supernatants were lyophilized and the frozen dried material was solubilized in distilled water to test the hyperglycemic activity. Approximately one sinus gland equivalent was injected into each test animal.
Gel chromatograph)' To compare the molecular weight of several hyperglycemic active fractions, gel chromatography on Sephadex G-50 (Superfine) was performed. At first, the column (0.6 x 140cm) was equilibrated with 10mM ammonium acetate (pH 8.5) and calibrated with RNase (13,000), insulin (5700), glucagon (3550), bacitracin (1411). The flow rate was 2.2 ml/hr and the fraction volume was 1 ml. The absorbance of each fraction was read at 230nm with a Zeiss spectrophotometer.
Animals and starting material Prawns were collected in the bay of Concarneau and kept in running sea water in the laboratory. They were selected according to the intermolt C stage and to the size (about 6 cm). To test the hyperglycemic activity, unfed and eyestalkless animals, from which eyestalks were cut off 18 hr before injections, were used. All the test fractions were injected (10#l/animal) into the abdominal muscles. The haemolymph (about 50 #l/animal) was collected 2 hr after the injection by section of the telson and uropods. After deproteinization of the blood samples with 0.5 M perchloric acid, the glucose concentration was estimated by the Godperid test (Boehringer, Mannheim). To study the overall distribution of the hyperglycemic active material in the eyestalk, different structures (medulla terminalis, medulla interna, medulla externa, MTGX-1 organ region, MTGX-2 organ region, medulla externa X organ, organ of Bellonci and sinus gland) were dissected and collected in distilled water in a micropotter. After homogenization and centrifugation in a Beckman Microfuge at 10,000rpm, the supernatants were recovered. Hyperglycemic activity was tested after injection of 1 organ equivalent for each structure. The protein content per structure was measured by the method of Lowry et al. (t951).
RESULTS
Localization of the hyperglycemic activity in the structures of the eyestalk As shown in Table 1, selective injections of eyestalk structures indicate a hyperglycemic activity in the extracts of the total eyestalk, total medulla terminalis (MT) and externa (ME), respectively in the regions of the medulla ganglionic X organs 1 (MTGX-1) and 2 ( M T G X - 2 ) and the neurohaemal sinus gland (SG). On the contrary, injections of extracts of the medulla externa X organ (MEX) and of the organ of Bellonci (OB) remain comparable to injections of sea water (controls).
Purification of the hyperglycemic factor (s) From eyestalks. The detection of C H H activity
Table 1. Localization of CHH activity in the eyestalk structures ol Palaemon
Control Total eyestalks Medulla terminalis Medulla interna Medulla externa MTGX-I MTGX-2 MEX OB SG
serrattgs
Protein per structure (l~g)
]Lg glucose ml haemolymph +_ SEM)
980 76 10.5 31 21 68 5.3 4.4 8.4
37 * 26(7) 235 ± 93 17) 190 * 75 (51 54 + 15 121 2411 ~ 40 (2) 22 + 5 (2) 167 k 45 (2) 5~~ + 17t31 40 ± 30(4~ 215 +__82 (5)
The extracts were prepared in a micropotter with distilled water and centrifuged in Beckman microfuge at 10,000 rpm. Between brackets: number of experiments in which animals were injected with 1 organ equivalent. MTGX-I, MTGX-2: neurosecretory cell-groups 1 and 2 of the medulla terminalis called medulla terminalis ganglionaris X organs. MEX: Neurosecretory cell group of the medulla externa called medulla externa-X organ. OB: organ of Bellonci; SG: sinus gland.
after the purification procedure of Kleinholz and Keller (1973) was performed by bioassays for hyperglycemia in the fractions of the preparative electrophoresis step. As shown in Fig. la, a very high hyperglycemic response was induced after the injections of gel fractions 7 and 8, corresponding to 2 eyestalk equivalents each. These hyperglycemic fractions show a r~ index between 0.55 and 0.60 determined on the electrophoretic migration of these 2 fractions in a polyacrylamide gel (Fig. lb, c). A lower hyperglycemic activity can be attributed to an electrophoretical fraction of a rm index 0.30 and to a r,, index 0.71. By combining the 2 hyperglycemic fractions (7 and 8) a total protein content of 8.5 mg protein was found but was probably largely overestimated due to the Folin reagent. The molecular weights of these 3 fractions were determined immediately using gel filtration on Sephadex G 50 (superfine). In the three cases, the main hyperglycemic activity eluted with a elution volume of between 26 and 28 ml thus corresponding to a molecular weight of 8000. From extirpated sinus glands. Following the first steps of the procedure of Keller and Wunderer (1978) and Keller (1981), consisting of desaltation on Sephadex G 10 and a preparative polyacrylamide gel electrophoresis from sinus gland material, lyophilized and only extracted immediately before electrophoresis, the main hyperglycemic activity can be related to a protein band with a r,, index between 0.58 and 0.60 (Fig. 2 a, b). A lower hyperglycemic activity was detected in the gel region corresponding to protein material with a r m value of 0.71. However, after carrying out comparable electrophoresis from the same extract of sinus glands, after its storage for a long period at - 20°C, a hyperglycemic activity was detected only in an electrophoretic fraction with an index of 0.71 (Fig. 3a, b). The hyperglycemia produced by this fraction was twice as high as the reaction provoked by the biologically active fraction with an rm index 0.58-0.60. When the fractions containing C H H were pooled the protein content determined was found to be 234 ~g.
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Molecular weights were estimated for the C H H active fraction with a r,, index 0.71, using gel filtration on Sephadex G 50 (Superfine) (Fig. 4) and compared with the elution pattern of freshly prepared sinus gland extracts (Fig. 5). F o r the fraction 0.71, the apparent molecular weight oscillates between 2000
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Dose-response relations of the hyperglycemic factors To compare the hyperglycemic activity of the two fractions described (rm 0.59 prepared by eyestalks extraction and rm 0.71 prepared by sinus gland extraction), these fractions were injected at different concentrations. In Table 2 a bioassay shows that the induced hyperglycemic responses are proportional to the calculated amount of injected hormone.
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DISCUSSION
The presence of the hyperglycemic activity in the eyestalk structures revealed by bioassays confirms our preliminary histo-immunological results in Palaemon serratus, using an antiserum against CHH from the crayfish Astacus leptodactylus (Van Wormhoudt et al., 1981). These results are also in agreement with the comparative immunocytochemical investigations of the crustacean hyperglycemic hormone of some decapod Crustacea by GorgelsKallen et al. (1982). An immunocytochemical reaction is present in a considerable portion of the sinus gland in the MTGX-2sinus gland nerve and in a number of neurosecretory cells in the MTGX-2. In this way, the induced hyperglycemic activity by the extracts from the MTGX-2 region and that from the sinus gland were expected, while the reactivity in the total extract of the medulla terminalis, as well as in the extract of the MTGX-1 region, is probably related to the pathway of the X organ-sinus gland tract containing CHH. The hyperglycemia provoked by the total extract of the medulla externa cannot be explained for the moment, since no positive cells for the anti-CHH serum were observed in the MEX neurosecretory cells of Palaemon serratus. Moreover,
this organ does not induce hyperglycemia (BellonHumbert et al., 1981). Purification of hyperglycemic material according to Kleinholz and Keller (1973) using total eyestalk extracts and according to Keller and Wunderer (1978) and to Keller (1981) using sinus gland material gives evidence for 3 forms of CHH. Yet, a major neuropeptide hormone with CHH activity and a rm index 0.59 in the polyacrylamide gel electrophoresis (7.5~) (Davis, 1964) is determined. This index represents an intermediary electrophoretic mobility between the indices of the Astacidae (r,, 0.35) and the Brachyura (r m 0.69) stated by Keller (1977). For the prawn Pandalus, a comparable r,, value 0.62 can be calculated from the results of Kleinholz and Keller (1973). The molecular weight estimation of this hyperglycemic fraction in the freshly prepared sinus gland material and after eyestalk purification allows us to determine its molecular weight at around 8000. Comparing these results on the hyperglycemic material from Palaemon serratus with related data in literature, we can deduce some homology with the results of Kleinholz and Keller (1973) who found an average of 6300 mol. wt for Pandalus, Skorkowski et al. (1977) with 7300 mol. wt in Crangon, Keller and Wunderer (1978) with 6726 mol. wt for Carcinus and
Crustacean hyperglycemic hormone
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Keller (1981) with 6812 mol. wt for Orconectes. However, after freezing and storage of the sinus gland extracts, the main highest hyperglycemic activity was correlated with an electrophoretical fraction with arm 0.71 and a molecular weight of 2000-3000. Some authors also reported hyperglycemic activity of small peptides at least in some conditions (Mordue and Stone, 1977; Carlsen et al., 1977; Loughton and Orchard, 1981). In Homarus, Trausch and Bauchau (1981) described small molecular peptides from eyestalks of about 1500 mol. wt with a hyperglycemic activity. This was obtained also after long storage and freezing and in the two cases they could correspond to partial hydrolysis of the main form of the hyperglycemic factor of 5000-7000 mol. wt recovered later in Homarus sinus glands (Van Deynen et al., 1983). Whether all these peptides are physiological can be studied in relation to prohormone and release studies in crustacean. In this way, the existence in the eyestalk of high molecular weight peptides referred by Skorkowki et al. (1977) and Andrew and Saleuddin (1979) should be reconsidered and also the existence of a polymorphism of the hormone as described
recently on reverse HPLC (Newcomb, 1983; Stuenkel, 1983). Comparing the 2 used procedures for purification, from the total eyestalk and from the sinus gland extracts, in relation to the protein recovery for CHH material, measured by the method of Lowry (1951), we find about 1 #g Lowry per eyestalk and 0.2/lg Lowry per sinus gland (between 0.5 and 1.3/lg according to different experiments). These results are
Table 2. Hyperglycemic effects of different concentrations of C H H extracts in Palaemon serratus ng protein (Lowry) injected
Eyestalk CHH r m 0.59
Sinus gland CHH rm 0.71
Control 50 500 5000
25 q: 9 38* 98* 230 :t: 30
62 q: 10 105 q: 20 170 q: 30 220 T 20
Blood glucose is expressed in #g/ml of haemolymph and corresponds to a pool of 2 x 5 prawns,* expected. Protein estimated by Lowry, largely overestimated C H H content (Keller, 1981).
360
A. VAN WORMHOUDT e l al.
in agreement with the d a t a from Skorkowski et al. (1977) for Crangon, w h o f o u n d a b o u t 1.2/~g Lowry per eyestalk a n d with the results of Keller and W u n d e r e r (1978) for Carcinus with 0.26 # g per sinus gland. The differences in recovery between the 2 procedures must be related to the p r o p e r characteristics of each purification step. F o r example, the recovery o f material from polyacrylamide gels is not always easy while dialysis of the eyestalk material was carried o u t with dialysis m e m b r a n e s with a cut off of 3500 mol. wt. Meanwhile, by R.I.A., Jaros a n d Keller (1979) have f o u n d approximately 1.2 # g of C H H in Carcinus sinus gland, using an a n t i s e r u m against Carcinus C H H . This a m o u n t is c o m p a r a b l e to the a m o u n t of 1 p g we have recovered per total eyestalk by the modified procedure of Kleinholz a n d Keller (1973), but is higher t h a n the value given for O r conectes (Keller, 1981) a r o u n d 0 . 0 3 # g Lowry per sinus gland. Fifty n a n o g r a m m e s of C H H (r,, 0.59 or r m 0.71) m e a s u r e d by Lowry are effective to stimulate at least twice the glycemia c o n t e n t in p r a w n s of 2 g b u t the exact value of pure C H H is not known. Lower values are o b t a i n e d for Carcinus (Keller a n d W u n d e r e r , 1977) or for Uca (Keller, 1981). This difference could be due to the instability of the molecule before and d u r i n g the injection or some seasonal influence (Leuven et al., 1982). REFERENCES
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