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Further characterization of growth hormone from the chum salmon (Oncorhynchus keta)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Further Characterization G. F. WAGNER,* *Department Department
of Physiology, of Biological
60, 27-34
(1985)
of Growth Hormone from the Chum Salmon (Oncorhynchus keta)’
R. C. FARGHER, University Sciences,
J. C. BROWN,*
AND B. A. MCKEOWN
of British Columbia, Vancouver, British Columbia, Canada; and Simon Fraser University, Burnaby, British Columbia, Canada
Accepted January 21, 1985 This report describes the isolation of growth hormone (GH) from the chum salmon (Onketa) pituitary using gel, afftnity, and ion exchange chromatography. Chum GH has an estimated molecular weight of 23,500 and an amino acid composition that is consistent with a vertebrate CH. The differentially charged forms of chum GH which are only apparent under alkaline conditions were separated by ion exchange and compared immunologically and biologically; Peak I, which consists of a single band (Rr = 0.35) under alkaline electrophoresis and Peak II which consists of two bands with R,‘s of 0.41 and 0.45. Both forms were found to be immunologically identical by immunodiffusion and to have similar growth promoting properties in intact rainbow trout (Salmo gairdneri). Chum GH was also active in the rat tibia test at a daily dosage of 70 l&animal. The results are discussed in relation to previous studies with chum GH and other fish GHs. B 1985 Academic corhynchus
Subsequent to the reported purification of chum salmon (Oncorhynchus keta) prolactin by Idler et al. in 1978, Komourdjian and Idler (1979) identified a second pituitary fraction which exhibited growth-promoting activity in hypophysectomized rainbow trout (Salmo gairdneri). This fraction, which they called a somatotropic principle, bound weakly to a DEAE anion exchanger at pH 9.0 and had an estimated molecular weight of 18,400. An antiserum raised against this substance, bound specifically to the growth hormone (GH) cells in the rainbow trout pituitary gland. It would appear therefore, that this somatotropic principle is similar to or synonymous with GH, on the basis of these findings. Due to our own interest in salmon GH physiology, we elected to further characterize the chum somatotropic principle. Our findings also suggest that this fraction is chum salmon GH, and in this report we have provided additional information on the
amino acid composition, electrophoretic characteristics, and biological activity of this hormone. MATERIALS
Purification
AND METHODS
of Chum Growth Hormone
Pituitary glands from chum (0. keta) salmon were collected between September and November (1981) with the cooperation of British Columbia Packers, Steveston, B.C. The purification protocol used was based on Idler et a/. (1978) with modifications. Routinely, 35 g of frozen pituitaries were homogenized in 70 ml of ice-cold 0.003 M Tris-acetate buffer, containing either 0.001 M phenylmethylsulfonylfluoride (Sigma) or 1000 kallikrein inhibitor units/ml of Trasylol (Boehringer-Mannheim) as protease inhibitors. The homogenate was adjusted to pH 8.5 with 1 N NaOH and stirred for 3 hr at 4”. The homogenate was centrifuged at 50,OOOg for 30 min and the supernatant was decanted and saved. This step was then repeated. The combined supernatants were applied to a calibrated 10 x 92 cm, Sephadex G-75 column and developed with homogenization buffer. The 18,5003 1,000 molecular weight fraction was saved (1500 ml) and passed through a column of concanavalin ASepharose (Con A) to remove the glycoproteins, principally gonadotropic and thyrotropic hormones. The sample was adjusted to the pH and ionic conditions of
t This work was presented in part at the Ninth International Symposium on Comparative Endocrinology, Hong Kong, 1981. 27
0016-6480185
$1.50
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
28
WAGNER
the Con A column before application (0.001 M MnCl,, 0.OO1MMgCI,,0.5MNaCI,0.04MTrisHCI,pH7.7). The Con A effluent containing unbound material was adjusted to 80% saturation with ammonium sulfate, stirred overnight at 4”, and centrifuged at 50,OOOg for 1 hr. The supematant was discarded and the pellet was dialyzed for 4 hr in 0.003 M Tris-acetate, pH 8.1. The dialyzate was centrifuged (SO,OOOg,Ii2 hr) and the supernatant applied to a calibrated, 5.0 x 92 cm, Sephadex G-75 column, equilibrated with 0.003 M T&-acetate, pH 8.1. The 18,500-31,000 molecular weight fraction was applied directly to a 1.6 x 50-cm column of DEAEBio-Gel (Bio-Rad) equilibrated with the same buffer. The column was flushed with several bed volumes of buffer before applying a linear gradient totalhng 600 ml (0.003 to 0.09 M Tris-acetate, pH 8.1). Chum GH eluted as two peaks, designated as Peak I (PI) and Peak II (PII). When analyzed electrophoretically at pH 8.3, PI consisted of a single band with an R, of 0.35, while PI1 consisted of two bands with R,‘s of 0.41 and 0.45. The separation between the two peaks was rarely complete so they were pooled, lyophihzed and desalted on a 3 x 85 cm Sephadex G100 column, equilibrated with 0.015 M ammonium acetate, pH 8.1. This final gel filtration also removed contaminating proteins of lower molecular weight which eluted at the trailing edge of PII. In one instance, however, chum salmon PI and PI1 were distinctly separated with no apparent cross contamination. In this case, the peaks were individually lyophilized and desalted as described earlier. Following the desalting step, the fractions containing GH were pooled and lyophilized. The final electrophoretic characteristics of chum salmon GH (PI and PI1 combined) were determined in disc gels at pH 8.3 (Omstein, 1964) and in acid-urea gels (Davis ef al., 1972). The molecular weight was estimated by sodium dodecyl sulfate (SDS) electrophoresis (Laemmli, 1970). In order to compare chum PI and PI1 immunologically, antibodies were raised against PI1 in New Zealand white rabbits. Animals were injected intradermally (Vaitukaitus et al., 1971) at approximately 30 sites with 25 kg of PII, once every month for a period of 4 months. The chum PII used for the immunization consisted of two bands, with R,‘s of 0.41 and 0.45, under alkaline (pH 8.3) electrophoresis. The rabbits were bled from an ear vein 10 days after each injection. The hormone was emulsified in Freund’s complete adjuvant for the first injection and incomplete adjuvant (Difco) thereafter. The specificity of this antiserum was assessed by immunocytochemistry in a manner similar to that described by Wagner and McKeown (1983) and was found to cross react specifically with the GH cells in the chum salmon pituitary gland. Furthermore, in immunodiffusion studies, a
ET AL. single precipitin line was observed when this antiserum was tested with a chum salmon pituitary extract (Wagner, 1984). Chum PI and PII were then compared for immunological similarities by Ouchterlony’s (1968) double gel diffusion technique.
Bioassay of Chum Growth Hormone Rainbow Trout
in
In the trout bioassay, chum salmon PI and PI1 were tested (double-blind) in intact, juvenile rainbow trout, S. gairdneri (5.34 k 1.3 g, X 4 SD, N = 40), to demonstrate their bioactivity and comparative growth-promoting abilities. Four groups of 10 fish were acclimated for 2 weeks in separate 20-liter glass aquaria, supplied with flow-through dechlorinated tap water (12”), and maintained on a 12D:12L photoperiod. The fish were then injected intraperitoneally, twice weekly with either chum PI, PII, or ovine GH (1 IU/mg, NIHGH-SlO) at a dosage of 1 pg/g body wt for 24 days. The fourth group, serving as a control, received equivalent dosages of bovine serum albumin (BSA). The injection vehicle consisted of 0.9% NaCl, pH 8.5. The fish were weighed prior to each injection and the dosages were adjusted accordingly. Oregon moist pellets (Moore-Clark) were provided to satiation twice daily. Growth was compared by calculating regression lines for the cumulative percentage weight gains of each group over the course of the experiment. The slopes were then compared by one-way analysis of variance and the Student-Newman-Keuls multiple range test. Slopes were considered to be significantly different if P < 0.05.
Bioassay of Chum Growth Hormone the Rat Tibia Test
in
Chum salmon GH (PI and PI1 combined) was assessed in the conventional mammalian GH bioassay, the rat tibia test (Greenspan et al., 1949). Male, Sprague-Dawley rats (100-120 g), which had been hypophysectomized at 28 days of age, were obtained from Charles River Ltd., Massachusetts. The rats were used 10 days postoperatively. Four groups of five rats received daily intraperitoneal injections of either: 70 p.g chum GH, 70 p.g ovine GH (1 IU/mg, NIH-GH-SlO), the equivalent of 5 mg of a sockeye (0. nerka) salmon pituitary extract, or 0.9% NaCI. The sockeye extract was prepared by homogenizing 0.5 g of pituitaries in 10 ml of ice-cold 0.9% NaCl. The homogenate was then adjusted to pH 8.5 with 1 N NaOH, stirred for 3 hr at 4”. and centrifuged at 20,OOOg for 30 min. The supematant was stored as frozen aliquots. Rats were injected daily with 100 pl of the supematant, representing the extract from 5 mg of pituitary glands. The hormones were dissolved in 0.9% NaCl, pH 8.5.
SALMON
GROWTH
Amino Acid Analysis of Chum Growth Hormone For the amino acid analysis of chum GH (PI and PII combined), three separate hydrolyses were performed on 50-kg aliquots of the same preparation, for 22 hr at 110’ under nitrogen. Amino acid analyses were performed on a Dionex (Sunnyvale, Calif.) 500B amino acid/peptide analyzer using the sodium “Hi-Phi” eluents. Five analyses were conducted on these hydrolyzates. Following each individual analysis, the computed ratios of each amino acid were converted to absolute values on the basis of 191 residues/molecule, the chain length of human GH. The values obtained from each analysis were then averaged to obtain the final amino acid composition. No correction was made for hydrolytic destruction.
RESULTS Purification
of Chum Growth Hormone
The final yield of the growth hormone from chum salmon pituitaries was l-5 mg/ 35 g. The yield was extremely variable, probably due to the poor condition of the glands upon arrival at the cannery. Of the two peaks eluting from DEAEBio-Gel (Fig. 1) which comprise chum
0.15
-
ABSORBANCE GRADIENT
10
20
29
HORMONE
salmon GH, Peak I was the more prevalent. The amount of Peak II which was obtained in the final yield varied between purifications. Chum GH eluted as a symmetrical peak on a Sephadex G-100 column (Fig. 2) with a K,, of 0.47. In alkaline (pH 8.3) gels, chum GH consisted of three bands with R,‘s of 0.35, 0.41, and 0.45 (Fig. 3A). In acid-urea gels (Fig. 3B), chum PI and PI1 migrated as single bands. In SDS gels (Fig. 3C), chum GH comigrated with the main band of ovine GH, with an estimated molecular weight of 23,500. Coho GH (Wagner and McKeown, 1984) which was included for comparison, migrated slightly ahead of chum GH with an estimated molecular weight of 22,500. In another of our chum GH preparations however, a contaminant was apparent following electrophoresis in acid-urea gels. This contaminant had a diffuse appearance in this gel system and migrated behind the GH band (Fig. 3D). In SDS gels, this contaminant appeared as two components, with molecular weights of 46,000 and
-
30
40
50 FRACTION
60
70
60
90
100
FIG. 1. Elution profile of the 18,500-31,000 molecular weight fraction of chum salmon pituitaries on DEAE-Bio-Gel. Growth hormone eluted between 0.025-0.045 M Tris-acetate as two peaks, I and II. This elution profile illustrates only the first 100 of a total of 117 fractions. In the last 17 fractions, two more protein peaks eluted, IV and V. respectively. Peaks III through V have not been characterized further.
WAGNER
30
ET AL.
03 r
FRACTION
FIG. 2. Elution profile of chum salmon growth hormone on a Sephadex G-100 column. Growth hormone eluted as a symmetrical peak with a VJV, ratio of 1.88 and a K,, of 0.41.
78,000, respectively. These contaminants may be higher-molecular-weight aggregates of GH, since they reappear after reelectrophoresis of GH eluted from an excised band. The immunodiffusion study of chum salmon PI and PII (Fig. 4) demonstrated that these proteins are immunologically identical; as indicated by the absence of spurs between the immunoprecipitin lines of the two antigens.
FIG. 4. Immunodiffusion of chum salmon Peak I (PI) and Peak II (PII) against an antiserum raised in rabbits to chum salmon PII. There are no spurs at the junctions of the two precipitin lines, indicating immunological identity.
The Bioassay of Chum Growth Hormone in Rainbow Trout
The results of the bioassay of chum salmon PI, PII, and ovine GH are shown in Fig. 5. When the slopes of the cumulative percentage weight gains over time were compared statistically, all hormone-treated groups had slopes which were greater than
100 -
.----.ChumPI .-.oGH . . . . . . ..Chum .-. BSA
t1
PII
60-
. 60.
40.
7.0. FIG. 3. Electrophoretograms of chum salmon growth hormone. (A) Alkaline (pH 8.3) electrophoresis (PI and PII combined). (B) Acid-urea electrophoresis of chum PI (left), a coho salmon rostra1 pars distalis extract (center) and chum PI1 (right). (C) Sodium dodecyl sulfate electrophoresis of chum GH (left), coho GH (middle), and ovine GH (right). (D) Acid-urea electrophoresis of a second preparation of chum GH showing a slower migrating contaminant. This contaminant consists of two components with estimated molecular weights of 46,000 and 78.000.
5
15
10
20
:
25
DAY
FIG. 5. The bioassay of chum salmon PI, PII, and ovine growth hormone in rainbow trout. All hormonetreated groups had greater slopes than the BSA-injetted control group (P < 0.001 in all cases). The slopes of the hormone-treated groups were also not significantly different from one another (P > 0.5 in all cases).
SALMON
GROWTH
TABLE 1 COMPARATIVE AMINO ACID COMPOSITIONOF CHUM SALMONANDCARPGROWTHHORMONEANDCHLJM SALMON PROLACTIN (PRL)
LYS His Arg Asx Thr Ser Glx Pro GUY Ala 'I2 cys Val Met Ile Leu Tyr Phe ‘b
Chum GH
Carp GH”
Chum PRLb
14.1 6.6 7.7 31.1 7.7 18.1 22.0 7.2 10.9 7.9 3.8 7.9 2.6 6.3 24.0 6.0 6.2 ND’
10.4 4.8 9.3 24.8 10.1 16.5 23.6 8.9 8.8 10.6 3.7 11.8 4.5 7.0 22.8 4.5 8.0 1.0
13.0 6.6 12.4 21.2 8.0 25.8 13.7 13.4 9.5 6.2 3.7 4.6 9.1 10.0 26.4 2.2 6.6 1.0
a Cook er al. (1983). b Kawauchi er al. (1983). c ND, not determined.
the control group (P < 0.001 in all cases), but not different than one another (P > 0.5 in all cases). The Bioassay of Chum Growth Hormone in the Rat Tibia Test All preparations induced significant tibia1 growth, when compared with the saline-injected control group (13.5 +- 5.3 urn, X 2 SEM). Ovine GH caused the largest growth increment (208.7 + 7.5 km, P < O.OOl), followed by chum GH (185.4 & 17.9 pm, P < 0.025), and the sockeye pituitary extract (172.9 t 10 pm, P < 0.01). One rat in the group injected with the pituitary extract had severe skeletal deformities by the end of the experiment and was excluded from the data analysis. Amino Acid Analysis of Chum Growth Hormone By amino acid analysis (Table I), chum GH was shown to have the salient charac-
31
HORMONE
teristics of a vertebrate GH; a low histidine and methionine content, the possibility of two disulphide bridges, and a high content of leucine and glutamic acid (Wilhelmi, 1974; Farmer and Papkoff, 1979). DISCUSSION
Despite the variable yields that were obtained using this purification protocol, GH was obtained in sufficient quantities for the purpose of bioassay and characterization. In SDS electrophoresis gels, chum GH migrated with a calculated molecular weight of 23,500 (Fig. 3). Chum PI and PI1 migrated as single bands in acid-urea electrophoresis gels, separately and when combined together. In alkaline (pH 8.3) gels, chum GH (PI and PI1 combined) separated into three distinct bands with R,‘s of 0.35, 0.41, and 0.45. PI consisted of a single band with an R, of 0.35, while PI1 contained the other two bands with R,‘s of 0.41 and 0.45. This behavior of chum GH in alkaline electrophoresis gels is also reflected in the elution sequence from DEAE-Bio-Gel (Fig. 2). The differing behavior of chum salmon GH in alkaline as opposed to acid gels has been observed for all vertebrate GHs, including all of the fish GHs that have been isolated thus far (Farmer et al., 1976, 1980; Cook et al., 1983). Several theories have been proffered as explanations, including N-terminal heterogeneity (Wallis and Davis, 1976), single base mutations on the N-terminal (Farmer et al., 1976), and deamidization (Lewis et al., 1970). Deamidization of glutamyl and asparaginyl residues occurs almost exclusively under alkaline conditions and is accelerated even further by high concentrations of ammonium sulfate (Lewis et al., 1970), both of which figured prominently in our purification procedure. Although we have not identified the specific cause for the various charged forms of chum GH, it is reasonable to suggest that deamidization may have taken place. In spite of the charge differences, chum salmon PI and PI1 appear to be similar.
32
WAGNER
ET AL.
They share immunological identity (Fig. 4) tained an estimated molecular weight of and they are both capable of promoting 23,500. However, this difference may only trout growth (Fig. 5) at a dosage of 1 be due to the different SDS procedures empg/g body wt. In an earlier reported isola- ployed. The fact that our chum GH comition of chum salmon GH, Idler et al. (1978) grated with the major component in ovine also identified two fractions eluting from GH indicates that our own estimate may be the ionic exchange column, DEAE 2 and in error. Li er al. (1973) have calculated the DEAE 3, each of which had growth-promolecular weight of ovine GH to be apmoting activity in rainbow trout. DEAE 2 proximately 22,000. Therefore, our molecwas more potent than DEAE 3 in increasing ular weight estimate may be 1500 too high. the growth rate of rainbow trout. It was On the basis of previous studies with fish concluded that DEAE 2 was growth hor- GHs, the tibia1 bioactivity displayed by mone and that the activity inherent in chum GH and the sockeye pituitary extract DEAE 3 was due to cross contamination was entirely unexpected. Pituitary extracts (Komourdjian and Idler, 1979). It appears and GHs purified from primitive fishes from our findings, however, that DEAE 2 (elasmobranchs, chondrosteans, holoscorresponds to what has been described teans, crossopterygians, and dipnoans) are here as PI and that DEAE 3 may be syn- all bioactive in the rat tibia test (Hayashida onymous with PII. and Lagios, 1969; Hayashida, 1970, 1971, Two GH species have also been identi1973; Hayashida and Lewis, 1978; Farmer fied in the eel (Anguilla anguilla) pituitary et al., 1980) as are GHs from representagland (Ingleton and Stribley, 1977). In this tives of all the higher vertebrate classes instance GHl and GH2, as they were clas- (Farmer and Papkoff, 1979). Until now, sified, were separated by alkaline electrohowever, teleost GHs and pituitary extracts phoresis (pH 8.3) and compared immunohave been found to possess little or no logically. Antisera raised against each GH bioactivity in this same assay (Hayashida species reacted specifically with the GH and Lagios, 1969; Farmer et al., 1976). Furcells in the eel pituitary and both GHs thermore, immunodiffusion and radioimshared immunological identity as well. It munoassay (RIA) studies employing a rat appears likely therefore, that the different GH antiserum have also demonstrated the molecular species which comprise a verteimmunological uniqueness of teleost GHs brate GH are biologically and immunolog(Hayashida and Lagios, 1969; Farmer et al., ically similar, despite the observed charge 1976; Hayashida and Lewis, 1978). The redifferences. sults of these previous studies have In future purifications of salmon GH, prompted Hayashida to conclude that the however, it may be advisable to use only teleosts are an evolutionary divergent the Peak I fraction for physiological group, at least with respect to the GH molstudies. If the charge differences of Peak II ecule. are due to damage of some kind (e.g., the There is also evidence of immunological diversity within the superorder Teleostei. poor condition of the pituitaries), this damage could result in subtle or even rad- Farmer et al. (1976) obtained only weak ical changes in the true physiology of the cross reactivity with a perch (Phanerodon hormone. furcatus) pituitary extract in a tilapia (SarOne notable difference between Idler et otherodon mossambicus) GH RIA. More real. (1978) and our findings is in the esticently, Cook er al. (1983) has demonstrated the poor immunoreactivity of coho salmon mated molecular weight of chum salmon GH. They found that chum GH had a mo- (0. kisutch) plasma GH in a homologous lecular weight of 18,400 whereas we ob- RIA for carp (Cyprinus carpio) GH. In view
SALMON
GROWTH
of these latter studies and the rat tibia test results in the present study, it appears that the teleost GHs are both biologically and immunologically diverse. The degree to which this diversity is phylogenically related, however, will only be forthcoming with further studies. To date, three fish growth hormones have been successfully isolated to homogeneity and characterized with respect to electrophoretic mobility, molecular weight, and amino acid composition; two from the superorder Teleostei (Farmer et al., 1976; Cook et al., 1983) and one from the superorder Chondrostei (Farmer et al., 1980). All of these fish GHs consist of three bands upon electrophoresis under alkaline conditions and one band under acidic conditions. The teleost GHs, tilapia and carp, have molecular weights of 22,200 and 22,500, respectively, whereas chondrostean (sturgeon) GH has a molecular weight of 23,500. By comparison, chum GH has similar electrophoretic characteristics and a similar estimated molecular weight. Comparatively, chum salmon GH is most similar in amino acid composition to carp GH (Table l), which also has equimolar concentrations of leucine, aspartic, and glutamic acid (Cook et al., 1983). In comparison to chum salmon prolactin (Kawauchi et al., 1983), chum salmon GH displays some major compositional differences (Table 1). Salmon GH has approximately twice the number of tyrosine, valine, and glutamic acid residues as salmon prolactin, in addition to lower quantities of serine, proline, arginine, and especially methionine. The two hormones are similar in most other respects. ACKNOWLEDGMENTS We thank Dr. Terry Owen and Syndel Laboratories, Vancouver for their assistance in the isolation procedures. We also gratefully acknowledge the cooperation of Mr. Ron Small, British Columbia Packers, in the collection of the salmon pituitary glands. This work was supported by an NSERC Grant (A9434) to
HORMONE
33
B.A.M. and British Columbia Research and Technology Awards to G.F. W. and R.C.F.
REFERENCES Cook, A. F., Wilson, S. W., and Peter, R. E. (1983). Development and validation of a carp growth hormone radioimmunoassay. Gen. Comp. Endocrinol. 50, 335-347. Davis, R. H., Copenhaver, J. H., and Carver, M. J. (1972). Characterization of acidic proteins in cell nuclei from rat brain by high resolution acrylamide gel electrophoresis. J. Neurochem. 19, 473-477. Ellis, S., Lorenson, M., Grindeland, R. E., and Callahan, P. K. (1972). Separation of the phenylalanyl and alanyl chains of bovine and ovine growth hormone by electrofocusing. In “Growth and Growth Hormone” (Pecile, A., and Muller, E. E., eds.), pp. 55-67, Excerpta Medica, Amsterdam. Farmer, S. W., Hayashida, T., Papkoff, H., and Polenov, A. L. (1980). Characteristics of growth hormone isolated from sturgeon (Acipenser guldenstadti) pituitaries. Endocrinology 108, 377381. Farmer, S. W., and Papkoff, H. (1979). Comparative biochemistry of pituitary growth hormone, prolactin and the glycoprotein hormones. In “Hormones and Evolution” (E. .I. W. Barrington, ed.), Vol. 2, pp 525-560, Academic Press, New York. Farmer, S. W., Papkoff, H., Hayashida, T., Bewley, T. A., Bern. H. A., and Li, C. H. (1976). Purilication and properties of teleost growth hormone. Gen. Comp. Endocrinol. 30, 91-100. Greenspan, F. S., Li, C. H., Simpson, M. E., and Evans, H. M. (1949). Bioassay of hypophyseal growth hormone: The tibia test. Endocrinology 45, 455-463. Hayashida, T. (1970). Immunological studies with rat pituitary growth hormone (RGH). II. Comparative immunochemical investigation of GH from representatives of various vertebrate classes with monkey antiserum to RGH. Gen. Comp. Endocrinol. 15, 432-452. Hayashida, T. (1971). Biological and immunochemical studies with growth hormone in pituitary extracts of holostean and chondrostean fishes. Gen. Comp. Endocrinol. 17, 275-280. Hayashida, T. (1973). Biological and immunochemical studies with growth hormone in pituitary extract of elasmobranchs. Gen. Comp. Endocrinol. 20, 377-385. Hayashida, T., and Lagios, M. D. (1969). Fish growth hormone: A biological, immunochemical, and ultrastructural study of sturgeon and paddlefish pituitaries. Gen. Comp. Endocrinol. 13, 403-411. Hayashida, T., and Lewis, U. J. (1978). Immunochem-
WAGNER
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ical and biological studies with antiserum to shark growth hormone. Gen. Comp. Endocrinol. 36, 530-542. Idler, D. R., Shamsuzzaman, K., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Gen. Comp. Endocrinol. 35, 409-418. Ingleton, P. M., and Stribley, M. F. (1977). Immunofluorescent identification of the cells of origin of eel (Anguilla anguilla) pituitary hormones separated by polyacrylamide gel electrophoresis. GPIZ. Comp. Endocrinol. 31, 37-44. Kawauchi, H., Ken-ichi, A., Takahashi, A., Hirano, T., Hasegawa, S., Naito, N., and Nakai, Y. (1983). Isolation and properties of chum salmon prolactin. Gen. Comp. Endocrinol. 49, 446-458. Komourdjian, M. P., and Idler, D. R. (1979). Chum salmon pituitary fractions: Somatotropic activity and immunocytofluorescence studies. Gen. Comp.
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Laemmli, U. K. (1970). Cleavage of structural proteins during the assembled bacteriophage T4. Nuture (London)
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Lewis, U. J., Cheever, E. V., and Hopkins, W. C. (1970). Kinetic study of the deamidation of growth hormone and prolactin. Biochim. Biophys. Acta 214, 498-508. Lewis, U. J.. Singh, R. N. P., Seavey, B. K., Lasker. R., and Pickford, G. E. (1972). Growth hormone and prolactin-like proteins of the blue shark (Prionace
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Li, C. H., Gordon, D., and Knorr, J. (1973). The primary structure of sheep pituitary growth hormone. Arch. Biochem. Biophys. 156, 493-508. Ornstein. L. (1964). Disc electrophoresis. 1. Background and theory. Ann. N.Y. Acud. Sci. 121, 321-349. Ouchterlony, 0. (1968). “Handbook of Immunodiffu-
ET AL. sion and lmmunoelectrophoresis.” Ann Arbor Science Publishers, Ann Arbor, Michigan. Vaitukaitus, J., Robbins. J. B., Nieschlag, E., and Ross, G. D. (1971). A method for producing specific antisera with small doses of immunogen. J. Clin.
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Wagner, G. F. (1985). “Studies on the Chemistry and Physiology of Salmon Growth Hormone.” Ph.D thesis, Simon Fraser University, British Columbia, Canada. Wagner, G. F., and McKeown, B. A. (1983). The immunocytochemical localization of pituitary somatotrops in the genus Oncorhynchus using an antiserum to growth hormone of chum salmon (Oncorhynchus keta). Cell Tissue Res. 231, 693697. Wagner, G. F., and McKeown, B. A. (1981):The purification, partial characterization and bioassay of growth hormone from two species of pacific salmon. In “Comparative Endocrinology” (B. Lofts, ed.), Proceedings of the 9th International Symposium on Comparative Endocrinology, Hong Kong 1981. University of Hong Kong Press, in press. Wallis. M.. and Davis, R. V. (1976). Studies on the chemistry of bovine and rat growth hormones. In “Growth Hormone and Related Peptides” (Petile. A. and Muller, E. E., eds.), pp. I- 13. Excerpts Medica, Amsterdam/Oxford. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determination by dodecylsulphate polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. Wilhelmi. A. E. (1974). Chemistry of growth hormone. In “Handbook of Physiology” (Knobil, E. and Sawyer, H., eds.), Sec. 7, Vol. IV, Part 2, American Physiological Society, Washington, D.C.
AND
COMPARATIVE
ENDOCRINOLOGY
Further Characterization G. F. WAGNER,* *Department Department
of Physiology, of Biological
60, 27-34
(1985)
of Growth Hormone from the Chum Salmon (Oncorhynchus keta)’
R. C. FARGHER, University Sciences,
J. C. BROWN,*
AND B. A. MCKEOWN
of British Columbia, Vancouver, British Columbia, Canada; and Simon Fraser University, Burnaby, British Columbia, Canada
Accepted January 21, 1985 This report describes the isolation of growth hormone (GH) from the chum salmon (Onketa) pituitary using gel, afftnity, and ion exchange chromatography. Chum GH has an estimated molecular weight of 23,500 and an amino acid composition that is consistent with a vertebrate CH. The differentially charged forms of chum GH which are only apparent under alkaline conditions were separated by ion exchange and compared immunologically and biologically; Peak I, which consists of a single band (Rr = 0.35) under alkaline electrophoresis and Peak II which consists of two bands with R,‘s of 0.41 and 0.45. Both forms were found to be immunologically identical by immunodiffusion and to have similar growth promoting properties in intact rainbow trout (Salmo gairdneri). Chum GH was also active in the rat tibia test at a daily dosage of 70 l&animal. The results are discussed in relation to previous studies with chum GH and other fish GHs. B 1985 Academic corhynchus
Subsequent to the reported purification of chum salmon (Oncorhynchus keta) prolactin by Idler et al. in 1978, Komourdjian and Idler (1979) identified a second pituitary fraction which exhibited growth-promoting activity in hypophysectomized rainbow trout (Salmo gairdneri). This fraction, which they called a somatotropic principle, bound weakly to a DEAE anion exchanger at pH 9.0 and had an estimated molecular weight of 18,400. An antiserum raised against this substance, bound specifically to the growth hormone (GH) cells in the rainbow trout pituitary gland. It would appear therefore, that this somatotropic principle is similar to or synonymous with GH, on the basis of these findings. Due to our own interest in salmon GH physiology, we elected to further characterize the chum somatotropic principle. Our findings also suggest that this fraction is chum salmon GH, and in this report we have provided additional information on the
amino acid composition, electrophoretic characteristics, and biological activity of this hormone. MATERIALS
Purification
AND METHODS
of Chum Growth Hormone
Pituitary glands from chum (0. keta) salmon were collected between September and November (1981) with the cooperation of British Columbia Packers, Steveston, B.C. The purification protocol used was based on Idler et a/. (1978) with modifications. Routinely, 35 g of frozen pituitaries were homogenized in 70 ml of ice-cold 0.003 M Tris-acetate buffer, containing either 0.001 M phenylmethylsulfonylfluoride (Sigma) or 1000 kallikrein inhibitor units/ml of Trasylol (Boehringer-Mannheim) as protease inhibitors. The homogenate was adjusted to pH 8.5 with 1 N NaOH and stirred for 3 hr at 4”. The homogenate was centrifuged at 50,OOOg for 30 min and the supernatant was decanted and saved. This step was then repeated. The combined supernatants were applied to a calibrated 10 x 92 cm, Sephadex G-75 column and developed with homogenization buffer. The 18,5003 1,000 molecular weight fraction was saved (1500 ml) and passed through a column of concanavalin ASepharose (Con A) to remove the glycoproteins, principally gonadotropic and thyrotropic hormones. The sample was adjusted to the pH and ionic conditions of
t This work was presented in part at the Ninth International Symposium on Comparative Endocrinology, Hong Kong, 1981. 27
0016-6480185
$1.50
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
28
WAGNER
the Con A column before application (0.001 M MnCl,, 0.OO1MMgCI,,0.5MNaCI,0.04MTrisHCI,pH7.7). The Con A effluent containing unbound material was adjusted to 80% saturation with ammonium sulfate, stirred overnight at 4”, and centrifuged at 50,OOOg for 1 hr. The supematant was discarded and the pellet was dialyzed for 4 hr in 0.003 M Tris-acetate, pH 8.1. The dialyzate was centrifuged (SO,OOOg,Ii2 hr) and the supernatant applied to a calibrated, 5.0 x 92 cm, Sephadex G-75 column, equilibrated with 0.003 M T&-acetate, pH 8.1. The 18,500-31,000 molecular weight fraction was applied directly to a 1.6 x 50-cm column of DEAEBio-Gel (Bio-Rad) equilibrated with the same buffer. The column was flushed with several bed volumes of buffer before applying a linear gradient totalhng 600 ml (0.003 to 0.09 M Tris-acetate, pH 8.1). Chum GH eluted as two peaks, designated as Peak I (PI) and Peak II (PII). When analyzed electrophoretically at pH 8.3, PI consisted of a single band with an R, of 0.35, while PI1 consisted of two bands with R,‘s of 0.41 and 0.45. The separation between the two peaks was rarely complete so they were pooled, lyophihzed and desalted on a 3 x 85 cm Sephadex G100 column, equilibrated with 0.015 M ammonium acetate, pH 8.1. This final gel filtration also removed contaminating proteins of lower molecular weight which eluted at the trailing edge of PII. In one instance, however, chum salmon PI and PI1 were distinctly separated with no apparent cross contamination. In this case, the peaks were individually lyophilized and desalted as described earlier. Following the desalting step, the fractions containing GH were pooled and lyophilized. The final electrophoretic characteristics of chum salmon GH (PI and PI1 combined) were determined in disc gels at pH 8.3 (Omstein, 1964) and in acid-urea gels (Davis ef al., 1972). The molecular weight was estimated by sodium dodecyl sulfate (SDS) electrophoresis (Laemmli, 1970). In order to compare chum PI and PI1 immunologically, antibodies were raised against PI1 in New Zealand white rabbits. Animals were injected intradermally (Vaitukaitus et al., 1971) at approximately 30 sites with 25 kg of PII, once every month for a period of 4 months. The chum PII used for the immunization consisted of two bands, with R,‘s of 0.41 and 0.45, under alkaline (pH 8.3) electrophoresis. The rabbits were bled from an ear vein 10 days after each injection. The hormone was emulsified in Freund’s complete adjuvant for the first injection and incomplete adjuvant (Difco) thereafter. The specificity of this antiserum was assessed by immunocytochemistry in a manner similar to that described by Wagner and McKeown (1983) and was found to cross react specifically with the GH cells in the chum salmon pituitary gland. Furthermore, in immunodiffusion studies, a
ET AL. single precipitin line was observed when this antiserum was tested with a chum salmon pituitary extract (Wagner, 1984). Chum PI and PII were then compared for immunological similarities by Ouchterlony’s (1968) double gel diffusion technique.
Bioassay of Chum Growth Hormone Rainbow Trout
in
In the trout bioassay, chum salmon PI and PI1 were tested (double-blind) in intact, juvenile rainbow trout, S. gairdneri (5.34 k 1.3 g, X 4 SD, N = 40), to demonstrate their bioactivity and comparative growth-promoting abilities. Four groups of 10 fish were acclimated for 2 weeks in separate 20-liter glass aquaria, supplied with flow-through dechlorinated tap water (12”), and maintained on a 12D:12L photoperiod. The fish were then injected intraperitoneally, twice weekly with either chum PI, PII, or ovine GH (1 IU/mg, NIHGH-SlO) at a dosage of 1 pg/g body wt for 24 days. The fourth group, serving as a control, received equivalent dosages of bovine serum albumin (BSA). The injection vehicle consisted of 0.9% NaCl, pH 8.5. The fish were weighed prior to each injection and the dosages were adjusted accordingly. Oregon moist pellets (Moore-Clark) were provided to satiation twice daily. Growth was compared by calculating regression lines for the cumulative percentage weight gains of each group over the course of the experiment. The slopes were then compared by one-way analysis of variance and the Student-Newman-Keuls multiple range test. Slopes were considered to be significantly different if P < 0.05.
Bioassay of Chum Growth Hormone the Rat Tibia Test
in
Chum salmon GH (PI and PI1 combined) was assessed in the conventional mammalian GH bioassay, the rat tibia test (Greenspan et al., 1949). Male, Sprague-Dawley rats (100-120 g), which had been hypophysectomized at 28 days of age, were obtained from Charles River Ltd., Massachusetts. The rats were used 10 days postoperatively. Four groups of five rats received daily intraperitoneal injections of either: 70 p.g chum GH, 70 p.g ovine GH (1 IU/mg, NIH-GH-SlO), the equivalent of 5 mg of a sockeye (0. nerka) salmon pituitary extract, or 0.9% NaCI. The sockeye extract was prepared by homogenizing 0.5 g of pituitaries in 10 ml of ice-cold 0.9% NaCl. The homogenate was then adjusted to pH 8.5 with 1 N NaOH, stirred for 3 hr at 4”. and centrifuged at 20,OOOg for 30 min. The supematant was stored as frozen aliquots. Rats were injected daily with 100 pl of the supematant, representing the extract from 5 mg of pituitary glands. The hormones were dissolved in 0.9% NaCl, pH 8.5.
SALMON
GROWTH
Amino Acid Analysis of Chum Growth Hormone For the amino acid analysis of chum GH (PI and PII combined), three separate hydrolyses were performed on 50-kg aliquots of the same preparation, for 22 hr at 110’ under nitrogen. Amino acid analyses were performed on a Dionex (Sunnyvale, Calif.) 500B amino acid/peptide analyzer using the sodium “Hi-Phi” eluents. Five analyses were conducted on these hydrolyzates. Following each individual analysis, the computed ratios of each amino acid were converted to absolute values on the basis of 191 residues/molecule, the chain length of human GH. The values obtained from each analysis were then averaged to obtain the final amino acid composition. No correction was made for hydrolytic destruction.
RESULTS Purification
of Chum Growth Hormone
The final yield of the growth hormone from chum salmon pituitaries was l-5 mg/ 35 g. The yield was extremely variable, probably due to the poor condition of the glands upon arrival at the cannery. Of the two peaks eluting from DEAEBio-Gel (Fig. 1) which comprise chum
0.15
-
ABSORBANCE GRADIENT
10
20
29
HORMONE
salmon GH, Peak I was the more prevalent. The amount of Peak II which was obtained in the final yield varied between purifications. Chum GH eluted as a symmetrical peak on a Sephadex G-100 column (Fig. 2) with a K,, of 0.47. In alkaline (pH 8.3) gels, chum GH consisted of three bands with R,‘s of 0.35, 0.41, and 0.45 (Fig. 3A). In acid-urea gels (Fig. 3B), chum PI and PI1 migrated as single bands. In SDS gels (Fig. 3C), chum GH comigrated with the main band of ovine GH, with an estimated molecular weight of 23,500. Coho GH (Wagner and McKeown, 1984) which was included for comparison, migrated slightly ahead of chum GH with an estimated molecular weight of 22,500. In another of our chum GH preparations however, a contaminant was apparent following electrophoresis in acid-urea gels. This contaminant had a diffuse appearance in this gel system and migrated behind the GH band (Fig. 3D). In SDS gels, this contaminant appeared as two components, with molecular weights of 46,000 and
-
30
40
50 FRACTION
60
70
60
90
100
FIG. 1. Elution profile of the 18,500-31,000 molecular weight fraction of chum salmon pituitaries on DEAE-Bio-Gel. Growth hormone eluted between 0.025-0.045 M Tris-acetate as two peaks, I and II. This elution profile illustrates only the first 100 of a total of 117 fractions. In the last 17 fractions, two more protein peaks eluted, IV and V. respectively. Peaks III through V have not been characterized further.
WAGNER
30
ET AL.
03 r
FRACTION
FIG. 2. Elution profile of chum salmon growth hormone on a Sephadex G-100 column. Growth hormone eluted as a symmetrical peak with a VJV, ratio of 1.88 and a K,, of 0.41.
78,000, respectively. These contaminants may be higher-molecular-weight aggregates of GH, since they reappear after reelectrophoresis of GH eluted from an excised band. The immunodiffusion study of chum salmon PI and PII (Fig. 4) demonstrated that these proteins are immunologically identical; as indicated by the absence of spurs between the immunoprecipitin lines of the two antigens.
FIG. 4. Immunodiffusion of chum salmon Peak I (PI) and Peak II (PII) against an antiserum raised in rabbits to chum salmon PII. There are no spurs at the junctions of the two precipitin lines, indicating immunological identity.
The Bioassay of Chum Growth Hormone in Rainbow Trout
The results of the bioassay of chum salmon PI, PII, and ovine GH are shown in Fig. 5. When the slopes of the cumulative percentage weight gains over time were compared statistically, all hormone-treated groups had slopes which were greater than
100 -
.----.ChumPI .-.oGH . . . . . . ..Chum .-. BSA
t1
PII
60-
. 60.
40.
7.0. FIG. 3. Electrophoretograms of chum salmon growth hormone. (A) Alkaline (pH 8.3) electrophoresis (PI and PII combined). (B) Acid-urea electrophoresis of chum PI (left), a coho salmon rostra1 pars distalis extract (center) and chum PI1 (right). (C) Sodium dodecyl sulfate electrophoresis of chum GH (left), coho GH (middle), and ovine GH (right). (D) Acid-urea electrophoresis of a second preparation of chum GH showing a slower migrating contaminant. This contaminant consists of two components with estimated molecular weights of 46,000 and 78.000.
5
15
10
20
:
25
DAY
FIG. 5. The bioassay of chum salmon PI, PII, and ovine growth hormone in rainbow trout. All hormonetreated groups had greater slopes than the BSA-injetted control group (P < 0.001 in all cases). The slopes of the hormone-treated groups were also not significantly different from one another (P > 0.5 in all cases).
SALMON
GROWTH
TABLE 1 COMPARATIVE AMINO ACID COMPOSITIONOF CHUM SALMONANDCARPGROWTHHORMONEANDCHLJM SALMON PROLACTIN (PRL)
LYS His Arg Asx Thr Ser Glx Pro GUY Ala 'I2 cys Val Met Ile Leu Tyr Phe ‘b
Chum GH
Carp GH”
Chum PRLb
14.1 6.6 7.7 31.1 7.7 18.1 22.0 7.2 10.9 7.9 3.8 7.9 2.6 6.3 24.0 6.0 6.2 ND’
10.4 4.8 9.3 24.8 10.1 16.5 23.6 8.9 8.8 10.6 3.7 11.8 4.5 7.0 22.8 4.5 8.0 1.0
13.0 6.6 12.4 21.2 8.0 25.8 13.7 13.4 9.5 6.2 3.7 4.6 9.1 10.0 26.4 2.2 6.6 1.0
a Cook er al. (1983). b Kawauchi er al. (1983). c ND, not determined.
the control group (P < 0.001 in all cases), but not different than one another (P > 0.5 in all cases). The Bioassay of Chum Growth Hormone in the Rat Tibia Test All preparations induced significant tibia1 growth, when compared with the saline-injected control group (13.5 +- 5.3 urn, X 2 SEM). Ovine GH caused the largest growth increment (208.7 + 7.5 km, P < O.OOl), followed by chum GH (185.4 & 17.9 pm, P < 0.025), and the sockeye pituitary extract (172.9 t 10 pm, P < 0.01). One rat in the group injected with the pituitary extract had severe skeletal deformities by the end of the experiment and was excluded from the data analysis. Amino Acid Analysis of Chum Growth Hormone By amino acid analysis (Table I), chum GH was shown to have the salient charac-
31
HORMONE
teristics of a vertebrate GH; a low histidine and methionine content, the possibility of two disulphide bridges, and a high content of leucine and glutamic acid (Wilhelmi, 1974; Farmer and Papkoff, 1979). DISCUSSION
Despite the variable yields that were obtained using this purification protocol, GH was obtained in sufficient quantities for the purpose of bioassay and characterization. In SDS electrophoresis gels, chum GH migrated with a calculated molecular weight of 23,500 (Fig. 3). Chum PI and PI1 migrated as single bands in acid-urea electrophoresis gels, separately and when combined together. In alkaline (pH 8.3) gels, chum GH (PI and PI1 combined) separated into three distinct bands with R,‘s of 0.35, 0.41, and 0.45. PI consisted of a single band with an R, of 0.35, while PI1 contained the other two bands with R,‘s of 0.41 and 0.45. This behavior of chum GH in alkaline electrophoresis gels is also reflected in the elution sequence from DEAE-Bio-Gel (Fig. 2). The differing behavior of chum salmon GH in alkaline as opposed to acid gels has been observed for all vertebrate GHs, including all of the fish GHs that have been isolated thus far (Farmer et al., 1976, 1980; Cook et al., 1983). Several theories have been proffered as explanations, including N-terminal heterogeneity (Wallis and Davis, 1976), single base mutations on the N-terminal (Farmer et al., 1976), and deamidization (Lewis et al., 1970). Deamidization of glutamyl and asparaginyl residues occurs almost exclusively under alkaline conditions and is accelerated even further by high concentrations of ammonium sulfate (Lewis et al., 1970), both of which figured prominently in our purification procedure. Although we have not identified the specific cause for the various charged forms of chum GH, it is reasonable to suggest that deamidization may have taken place. In spite of the charge differences, chum salmon PI and PI1 appear to be similar.
32
WAGNER
ET AL.
They share immunological identity (Fig. 4) tained an estimated molecular weight of and they are both capable of promoting 23,500. However, this difference may only trout growth (Fig. 5) at a dosage of 1 be due to the different SDS procedures empg/g body wt. In an earlier reported isola- ployed. The fact that our chum GH comition of chum salmon GH, Idler et al. (1978) grated with the major component in ovine also identified two fractions eluting from GH indicates that our own estimate may be the ionic exchange column, DEAE 2 and in error. Li er al. (1973) have calculated the DEAE 3, each of which had growth-promolecular weight of ovine GH to be apmoting activity in rainbow trout. DEAE 2 proximately 22,000. Therefore, our molecwas more potent than DEAE 3 in increasing ular weight estimate may be 1500 too high. the growth rate of rainbow trout. It was On the basis of previous studies with fish concluded that DEAE 2 was growth hor- GHs, the tibia1 bioactivity displayed by mone and that the activity inherent in chum GH and the sockeye pituitary extract DEAE 3 was due to cross contamination was entirely unexpected. Pituitary extracts (Komourdjian and Idler, 1979). It appears and GHs purified from primitive fishes from our findings, however, that DEAE 2 (elasmobranchs, chondrosteans, holoscorresponds to what has been described teans, crossopterygians, and dipnoans) are here as PI and that DEAE 3 may be syn- all bioactive in the rat tibia test (Hayashida onymous with PII. and Lagios, 1969; Hayashida, 1970, 1971, Two GH species have also been identi1973; Hayashida and Lewis, 1978; Farmer fied in the eel (Anguilla anguilla) pituitary et al., 1980) as are GHs from representagland (Ingleton and Stribley, 1977). In this tives of all the higher vertebrate classes instance GHl and GH2, as they were clas- (Farmer and Papkoff, 1979). Until now, sified, were separated by alkaline electrohowever, teleost GHs and pituitary extracts phoresis (pH 8.3) and compared immunohave been found to possess little or no logically. Antisera raised against each GH bioactivity in this same assay (Hayashida species reacted specifically with the GH and Lagios, 1969; Farmer et al., 1976). Furcells in the eel pituitary and both GHs thermore, immunodiffusion and radioimshared immunological identity as well. It munoassay (RIA) studies employing a rat appears likely therefore, that the different GH antiserum have also demonstrated the molecular species which comprise a verteimmunological uniqueness of teleost GHs brate GH are biologically and immunolog(Hayashida and Lagios, 1969; Farmer et al., ically similar, despite the observed charge 1976; Hayashida and Lewis, 1978). The redifferences. sults of these previous studies have In future purifications of salmon GH, prompted Hayashida to conclude that the however, it may be advisable to use only teleosts are an evolutionary divergent the Peak I fraction for physiological group, at least with respect to the GH molstudies. If the charge differences of Peak II ecule. are due to damage of some kind (e.g., the There is also evidence of immunological diversity within the superorder Teleostei. poor condition of the pituitaries), this damage could result in subtle or even rad- Farmer et al. (1976) obtained only weak ical changes in the true physiology of the cross reactivity with a perch (Phanerodon hormone. furcatus) pituitary extract in a tilapia (SarOne notable difference between Idler et otherodon mossambicus) GH RIA. More real. (1978) and our findings is in the esticently, Cook er al. (1983) has demonstrated the poor immunoreactivity of coho salmon mated molecular weight of chum salmon GH. They found that chum GH had a mo- (0. kisutch) plasma GH in a homologous lecular weight of 18,400 whereas we ob- RIA for carp (Cyprinus carpio) GH. In view
SALMON
GROWTH
of these latter studies and the rat tibia test results in the present study, it appears that the teleost GHs are both biologically and immunologically diverse. The degree to which this diversity is phylogenically related, however, will only be forthcoming with further studies. To date, three fish growth hormones have been successfully isolated to homogeneity and characterized with respect to electrophoretic mobility, molecular weight, and amino acid composition; two from the superorder Teleostei (Farmer et al., 1976; Cook et al., 1983) and one from the superorder Chondrostei (Farmer et al., 1980). All of these fish GHs consist of three bands upon electrophoresis under alkaline conditions and one band under acidic conditions. The teleost GHs, tilapia and carp, have molecular weights of 22,200 and 22,500, respectively, whereas chondrostean (sturgeon) GH has a molecular weight of 23,500. By comparison, chum GH has similar electrophoretic characteristics and a similar estimated molecular weight. Comparatively, chum salmon GH is most similar in amino acid composition to carp GH (Table l), which also has equimolar concentrations of leucine, aspartic, and glutamic acid (Cook et al., 1983). In comparison to chum salmon prolactin (Kawauchi et al., 1983), chum salmon GH displays some major compositional differences (Table 1). Salmon GH has approximately twice the number of tyrosine, valine, and glutamic acid residues as salmon prolactin, in addition to lower quantities of serine, proline, arginine, and especially methionine. The two hormones are similar in most other respects. ACKNOWLEDGMENTS We thank Dr. Terry Owen and Syndel Laboratories, Vancouver for their assistance in the isolation procedures. We also gratefully acknowledge the cooperation of Mr. Ron Small, British Columbia Packers, in the collection of the salmon pituitary glands. This work was supported by an NSERC Grant (A9434) to
HORMONE
33
B.A.M. and British Columbia Research and Technology Awards to G.F. W. and R.C.F.
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ical and biological studies with antiserum to shark growth hormone. Gen. Comp. Endocrinol. 36, 530-542. Idler, D. R., Shamsuzzaman, K., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Gen. Comp. Endocrinol. 35, 409-418. Ingleton, P. M., and Stribley, M. F. (1977). Immunofluorescent identification of the cells of origin of eel (Anguilla anguilla) pituitary hormones separated by polyacrylamide gel electrophoresis. GPIZ. Comp. Endocrinol. 31, 37-44. Kawauchi, H., Ken-ichi, A., Takahashi, A., Hirano, T., Hasegawa, S., Naito, N., and Nakai, Y. (1983). Isolation and properties of chum salmon prolactin. Gen. Comp. Endocrinol. 49, 446-458. Komourdjian, M. P., and Idler, D. R. (1979). Chum salmon pituitary fractions: Somatotropic activity and immunocytofluorescence studies. Gen. Comp.
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Li, C. H., Gordon, D., and Knorr, J. (1973). The primary structure of sheep pituitary growth hormone. Arch. Biochem. Biophys. 156, 493-508. Ornstein. L. (1964). Disc electrophoresis. 1. Background and theory. Ann. N.Y. Acud. Sci. 121, 321-349. Ouchterlony, 0. (1968). “Handbook of Immunodiffu-
ET AL. sion and lmmunoelectrophoresis.” Ann Arbor Science Publishers, Ann Arbor, Michigan. Vaitukaitus, J., Robbins. J. B., Nieschlag, E., and Ross, G. D. (1971). A method for producing specific antisera with small doses of immunogen. J. Clin.
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Wagner, G. F. (1985). “Studies on the Chemistry and Physiology of Salmon Growth Hormone.” Ph.D thesis, Simon Fraser University, British Columbia, Canada. Wagner, G. F., and McKeown, B. A. (1983). The immunocytochemical localization of pituitary somatotrops in the genus Oncorhynchus using an antiserum to growth hormone of chum salmon (Oncorhynchus keta). Cell Tissue Res. 231, 693697. Wagner, G. F., and McKeown, B. A. (1981):The purification, partial characterization and bioassay of growth hormone from two species of pacific salmon. In “Comparative Endocrinology” (B. Lofts, ed.), Proceedings of the 9th International Symposium on Comparative Endocrinology, Hong Kong 1981. University of Hong Kong Press, in press. Wallis. M.. and Davis, R. V. (1976). Studies on the chemistry of bovine and rat growth hormones. In “Growth Hormone and Related Peptides” (Petile. A. and Muller, E. E., eds.), pp. I- 13. Excerpts Medica, Amsterdam/Oxford. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determination by dodecylsulphate polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. Wilhelmi. A. E. (1974). Chemistry of growth hormone. In “Handbook of Physiology” (Knobil, E. and Sawyer, H., eds.), Sec. 7, Vol. IV, Part 2, American Physiological Society, Washington, D.C.