GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Purification
30,
91-100 (1976)
and Properties
of Teleost Growth Hormone
SUSAN WALKER FARMER, HAROLD PAPKOFF, TED HAYASHIDA,’ THOMAS A. BEWLEY,HOWARD A. BERN,~ AND CHOH HAO LI The Hormone 2Department
Research
Laboratory and ‘Department of Anatomy, University of California, San Francisco, California 94143 and of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720
Accepted May 7, 1976 Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedtmentatton coeffictent (s~~,~) of 2.19, and an a-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.
The presence of a growth hormone (GH) in fish pituitaries has been clearly established. Hypophysectomy of fish causes a cessation of growth, and replacement therapy with either fish pituitary extracts or mammalian GH causes a resumption of growth (Pickford and Atz, 1957). However, attempts by several workers to test pituitary extracts of modem bony fish (teleost) in the rat tibia assay have given negative results (Solomon and Greep, 1959; Moudgal and Li, 1961; Hayashida, 1970). Interestingly, pituitary extracts of primitive bony fish (Hayashida and Lagios, 1969), holosteans (Hayashida, 1971), and elasmobranchs (Hayashida, 1973), were shown to be active in the rat tibia assay. Similarly, when pituitary extracts of these fish were tested for cross-reaction in a rat GH radioimmunoassay, those of teleosts showed no appreciable immunochemical relatedness to
mammalian GH, while those of primitive bony fishes, holosteans, and elasmobranchs showed low but significant cross-reaction with the rat GH antiserum (Hayashida, 1970). These findings, coupled with the recently available comparative information on purified GHs from other nonmammalian species (avian, reptilian, and amphibian; Farmer et al., 1974,1976a), make a study of purified fish GH of special interest. However, attempts to purify fish GH have been severely hindered by the lack of a sensitive and convenient GH assay in fish, the inactivity of crude teleost pituitary extracts in the tibia assay, and the difficulty in obtaining sufficient quantities of fish pituitaries. Wilhelmi (1955) described two preparations of GH, from hake and pollack pituitaries, which stimulated growth in fish (Pickford, 1954) but were inactive in the rat tibia as91
Copyright @ 1976 by Academic Press. Inc. AI! rights of reproduction in any form reserved.
92
FARMER
say. Lewis et al. (1972), using disc electrophoresis for identification, purified a “GH-like protein” from shark pituitaries which was also found to stimulate growth in teleost fish. This report presents data on the isolation and characterization of highly purified GH teleost, Tilapia from a euryhaline mossambica.3 During our isolation of prolactin (PRL) from this species (Farmer et al., 1975, 1976b) all fractions were examined by disc gel electrophoresis. A side fraction from this purification was found to give a pattern that has been characteristic for mammalian GHs (Cheever and Lewis, 1969). This fraction was purified and chemically characterized. Activity in the rat tibia assay confirmed the identity of this material as teleost GH. MATERIALS
AND METHODS
Pituitaries. Mature Tilapia mossambica of both sexes were collected in Wahiawa Reservoir, Hawaii; the pituitaries were removed immediately, frozen, and kept at -20” until fractionation. A total of 20,001 pituitaries were obtained which amounted to 63 g wet weight. Fractionation. Fractionation methodology was similar to that used in our laboratory for the purification of GH from mammalian and nonmammalian species (Papkoff and Li, 1958; Papkoff et al., 1%2; Farmer et al., 1974, 1976a). The initial purification steps were also identical to those reported for the isolation of Tilapia PRL (Farmer et al., 1975) up to the point where PRL and GH were separated. In brief, the pituitaries were homogenized in a Waring Blendor with cold distilled water, adjusted to pH 9.5 with Ca(OH),, and stirred at 4” for 3 hr. After centrifugation, the alkaline extract was dialyzed, lyophilized, and then dissolved in phosphate buffer (pH 5.1), containing 12% saturated (NH&SO, for chromatography on Amberlite CG-50 as previously described (Papkoff et al., 1962). The fraction eluting with pH 6.0 buffer was shown to contain both the PRL and GH. Subsequent chromatography on DEAEcellulose, equilibrated with 0.03 M NH.,HCO,, pH 9, effected a separation of the PRL and GH; PRL was unadsorbed (Farmer et al., 1975) and GH was adsorbed and eluted with 0.2 M NKHCO,.
3 This Sarotherodon
species
has
mossambicus.
recently
been
renamed
ET AL.
Further purification of Tilapia GH was obtained by a series of precipitations at various pH values and salt and alcohol concentrations: Tilapia GH precipitated at pH 5.0; at pH 5.3 in the presence of 20% ethanol; at pH 3.5 adjusted with HPO, in the presence of 0.15 M (NH&SO,; and in 40% saturated (NH&SO,. In each case impurities remained in the supematant. Final purification was obtained by gel filtration on Sephadex G-100 equilibrated with 0.05 M NH4HC03. The material eluting with a VJVO of 2.1 was used for characterization after refiltration. Chemical and physical characterization. Pertinent fractions were examined by disc gel electrophoresis at pH 4.5 (Reisfeld et a/., 1962) and pH 8.3 (Omstein, 1964) in 7.5% acrylamide gels stained with amido schwarz. NH,-terminal analyses were performed by the dansyl technique (Gray, 1967; Woods and Wang, 1967); NH,-terminal sequence by the dansyl-Edman procedure (Gray, 1967; Woods and Wang, 1%7; Edman, 1950); COOH-terminal analyses by the carboxypeptidase reaction as previously described (Farmer et al., 1976a) and by hydrazinolysis (Niu and Fraenkel-Conrat, 1955); and amino acid analyses by the method of Spackman et al. (1958) in an automatic amino acid analyzer (Beckman Model 120B). Sedimentation studies on the Tilapia GH were performed in a Spinco Model E ultracentrifuge at a speed of 59,780 rpm, at room temperature. The hormone was dissolved in 0.1 M Tris-HCI, pH 8.2, at a concentration of 1 mg/ml. The molecular weight of Tilapia GH was estimated by exclusion chromatography on a column of Sephadex G-100 (1.5 x 59 cm) in 0.1 M Tris-HC1 buffer (pH 8.2). The sample was applied to the bottom of the column and eluted upward at a flow rate of 4.9 ml/hr using an LKB Model 10200 peristaltic pump. Elution patterns were continuously monitored near 280 nm with an LKB Uvicord-II. The column was calibrated with blue dextran 2000 (Pharmacia), myoglobin, bovine serum albumin (Miles Research Laboratories), and highly purified human GH. Stokes radii were calculated from the elution patterns as described by Laurent and Killander (1964). Circular dichroism (CD) spectra were recorded on a Cary Model 60 spectropolarimeter equipped with a Model 6002 circular dichroism attachment according to procedures previously outlined (Bewley et al., 1972). The content of a-helix was estimated as described elsewhere (Bewley et at., 1969). Protein concentrations were determined from absorption spectra taken in the Tris-HCl buffer on a Beckman DK-2A spectrophotometer. Spectra were recorded from 360 to 245 nm and corrected for light scattering as described by Beaven and Holiday (1952). An absorptivity value, E, c,,,,*,, n,“.l% = 0.975, was determined from the spectrum of a carefully weighed sample of Tilapia GH dissolved in the Tris-HCl buffer (pH 8.2),
TELEOST
GROWTH
after correcting for light scattering and assuming a 15% moisture content for the lyophilized protein. Bioassay. The Tilapia GH was tested for biological activity in the rat tibia assay (Greenspan ef al., 1949). Female Long-Evans rats were hypophysectomized at 28 days of age and used I days postoperatively for the assay. Highly purified bovine GH prepared in this laboratory (Li, 1954) was used as a standard. Immunochemistry. The immunochemical reactivity of Tilupia GH was tested with rat GH antiserum (Hayashida and Contopoulos, 1967) and snapping turtle GH antiserum (Hayashida et al., 1975), in double antibody radioimmunoassay systems. The techniques employed in the radioimmunoassays have been previously described (Hayashida, 1971). Purified rat GH (3.0 USP Ulmg; Ellis et al., 1968) was labeled with lz51 (Greenwood et al., 1963) and was also employed as standard. The second antibody used for the immunoprecipitation of the labeled rat GH was rabbit anti-human y-globulin serum. In addition, antiserum was produced against the Tilapia GH. A single young albino rabbit was immunized with a total of 4.5 mg of Tilupia GH. Four injections of 1 mg each were given in complete Freund’s adjuvant at weekly intervals. Each injection was divided into three equal portions, two being given SCand one ip. A booster injection of 0.5 mg was given in saline 10 days after the last weekly injection. The animal was bled 1 week after the booster injection and tested for the presence of precipitating antibodies by the Ouchterlony agar diffusion technique (Ouchterlony, 1953). The results were positive, and the animal was bled completely and sacrificed. Merthiolate (0.01%) was added to the serum as a preservative. The agar diffusion test with the antiserum revealed no precipitin line with Tilapia serum but a faint line with Tilapia PRL. Therefore the antiserum was absorbed with Tilapia PRL, as described by Hayashida (1970). After absorption, no precipitin line was observed with Tilapia PRL. The absorbed antiserum was used at a final dilution of 1:30,000 in a double antibody radioimmunoassay system, similar to that previously described (Hayashida, 1971). Tilapia GH was iodinated with rZsI by the method of Greenwood ef al. (1963) and was also employed as the standard.
RESULTS Chemical and Physical Characterization The Tilapia GH behaved identically to
known mammalian and nonmammalian GHs during fractionation, and a yield of 90 mg was obtained, which represents 1400 mg/kg wet weight tissue. This yield is comparable to the value of 2000 mg/kg obtained
HORMONE
93
for bovine GH (Li, 1954). The final purification step was gel filtration on Sephadex G-100, and the Tilapia GH emerged as a single symmetrical peak with a VJV,, of 2.1. This GH also showed a single symmetrical sedimenting boundary in the ultracentrifuge and a sedimentation coefficient (sz,& of 2.19 was calculated. Exclusion chromatography on a calibrated Sephadex G-100 column was performed to determine the molecular weight of the Tilapia GH. The GH exhibited a single, symmetrical peak with a V$V, ratio of 2.09. A Stokes radius of 22.9 A and a molecular weight of 22,200 daltons were calculated for Tilapia GH from its elution characteristics. These values may be compared with a VJV, of 2.10, a Stokes radius of 22.6 A, and a molecular weight of 22,100 daltons obtained for human GH monomer on this column (Bewley, unpublished results). During the purification of the Tilapia GH, the presumptive GH fraction was identified by polyacrylamide disc gel electrophoresis. The pattern obtained with the purified Tilapia GH at pH 8.3, shown in Fig. 1, has a major heavily stained band and two faster-moving, less intensely staining bands. An identical pattern has been found with representative species in each class of vertebrates (Farmer et al., 1976a). The two faster-moving bands may be deamidated forms of GH (Cheever and Lewis, 1969) or a reflection of microheterogeneity at the NH,-terminal residue (Lorenson and Ellis, 1975). The latter possibility was demonstrated to be true in the case of the Tilapia GH (see below). The fact that these additional bands are not indicative of the presence of other proteins is supported by the finding of a single band with this GH at pH 4.5 (Fig. 1). The observation that stained bands were not seen in other regions of the gel also indicates the absence of contaminating proteins. NH,-terminal analysis by the dansyl procedure showed the presence of isoleucine,
94
FARMER
ET AL.
TABLE AMINO
Amino acid LYS His Arg Asp Thr Ser Glu pro
GUY Ala ‘h-cys Val Met Ile Leu W Phe Trp
FIG. 1. Disc electrophoretic pattern of Tilapia GH at pH 4.5 (left) and pH 8.3 (right), 50 pg each on 7.5% gels stained with amido schwarz.
leucine, and valine in approximate amounts of 70, 20, and lo%, respectively. However, application of the dansyl-Edman procedure showed only a single residue, proline, at the next position. At the carboxyl terminal, a single residue, leucine, was found both by the hydrazinolysis reaction and by liberation with carboxypeptidase. These results suggest that the variation seen at the NH, terminus may represent single base mutations of the same basic molecule. The amino acid analysis of Tilapia GH is shown in Table 1, with that of ovine GH included for comparison. Because Tilapia GH was found to have a molecular weight similar to that of ovine GH, the amino acid analysis was calculated on the basis of Tilapia GH having the same number of res-
1
ACID COMPOSITION OF Tilapia GROWTH HORMONES COMPARED WITH OVINE GROWTH HORMONES
Ovine growth hormone 11 3 13 16 12 13 24 6 10 15 4 6 4 7 27 6 13 1
Tilapia
growth hormone 8.2 5.0 11.0 19.3 12.0 21.4 29.1 6.8 7.4 8.2 4.6 6.0 1.2 9.0 21.2 7.2 6.7 1.0
a Amino acid analysis: 20-hr hydrolysis, not corrected for hydrolytic destruction, calculated on the basis of 191 residues/mole; cystine and methionine values were obtained with performic acid-oxidized preparations (Li, 1957); the tryptophan value was determined by methane sulfonic acid hydrolysis (Moore, 1972) and by spectrophotometric measurement (Beaven and Holiday, 1952). All values are residues per mole. JJTaken from structural analysis of ovine GH, 191 residues (Li et al., 1973).
idues as ovine GH: 191 (Li et al., 1973). The Tilapia GH exhibits the features that have been found to characterize mammalian GHs, namely, two disulfides, a single tryptophan, low methionine and histidine content, and a high glutamic acid and leucine content (Wilhelmi, 1975). However, there are also notable differences between the two species: Tilapia GH has a high aspartic acid and serine content and a low methionine, and phenylalanine alanine, content compared with ovine GH. The circular dichroism spectra obtained with Tilapia GH are shown in Fig. 2. In the
TELEOST
GROWTH t12.5 -i
95
HORMONE T
B
0
d -‘2.5 1 -25.0 p -37.5 “5 -50.0 3
-625
-
-75.0 260
270
280
6 290
300
310
WAVELENGTH (NM)
FIG. 2. Circular dichroism spectra of Tikzpia GH in 0.1 M Tris-HCI buffer (pH 8.2), in the region of amide bond absorption (A) and in the region of side-chain absorption (B).
region dominated by amide bond absorption, the CD spectrum of Tilupia GH shows the two negative bands at 209 and 221 nm which are characteristic of the o-helix. To the nearest 5%, an a-helix content of 50% may be estimated for Tilupia GH from the ellipticities at either 209 or 221 nm. In the region of side-chain absorption, the CD spectrum of Tilapia GH exhibits a weak, positive band near 299 nm, two stronger, negative bands at 292 and 285 nm, a negative shoulder around 280 nm, a weak negative band at 269 nm, and a weak negative shoulder between 260 and 265 nm. Biological Characterization
The purified Tilapia GH was tested in the commonly employed mammalian growth hormone bioassay, the tibia test (Greenspan et al., 1949). It gave a low but significant response which was dose related, although not parallel to the bovine GH response (Table 2). The Tilupia GH TABLE Assay
2
OF BOVINE AND Tilapia BY THE TIBIA TEST
Preparation
Dose OLP)
Number of animals
Saline Bovine GH Bovine GH Tilapia GH Tilupia GH
20 60 400 1200
4 4 4 4 4
GH
Tibia width (wm k SEM) 181 k 199 !I 258 f 200 k 220 2
4 3 6 9 8
was also tested in two fish PRL bioassays. In the sodium-retention assay in Tilupia (Clarke, 1973), the Tilapia GH was inactive at a dose of 10 pg, and it was inactive at a dose of 50 pg in the Gillichthys urinary bladder assay (Doneen and Bern, 1974). Tilapia PRL was active in the same assays at doses of 0.1 and 1 pg, respectively (Farmer et al., 1976b). Immunochemical Characterization
The Tilapia GH was tested for immunochemical reactivity in two radioimmunoassay systems, employing rat GH antiserum (Hayashida, 1970) and snapping turtle GH antiserum (Hayashida et al., 1975). Pituitary extracts of teleost fish previously tested had shown extremely weak or no cross-reaction in these assays. Puritied Tilapia GH, however, significantly inhibited the binding of labeled rat GH in both assay systems (Fig. 3), although relatively large quantities of the hormone were required. The inhibition slope for Tilupia GH was parallel to that of the rat GH standard in the turtle GH antiserum system, but it was not quite parallel to the standard in the rat GH antiserum system. In preliminary studies the Tilapia GH antiserum was evaluated for specificity and cross-immunoreactivity. Shown in Fig. 4 is an Ouchterlony agar diffusion plate with Tilupia GH antiserum in the center well and various antigens in the peripheral wells. A
96
FARMER
:,
0
III/ .5 1 2 4 NANOGRAMS
I 16
I 64
I 250
I loo0
I , AC03 15,000
OF PURIFIED HORMONE
FIG. 3. Cross-reaction of purified Tilapiu GH in two different radioimmunoassays, one employing monkey antiserum to rat GH (solid lines) and the other employing monkey antiserum to snapping turtle GH (dashed lines). Highly purified rat GH (Ellis, 3.0 USP U/mg) was iodinated with 9 according to the method of Greenwood et al. (1%3) and was also used for the standard. All points are the averages of two determinations.
FIG. 4. Ouchterlony plate showing the reaction of unabsorbed rabbit serum to Tilapia GH (center well, 0.15 ml) with purified i”i~upia GH (10 pg), perch pituitary extract, sturgeon pituitary extract, rat GH (10 CLp), Tilapia PRL (20 pg), and normal Tilapiu serum (0.05 ml, 1:lO dilution). The pituitary extracts are the equivalent of 1.O mg of wet tissue weight/well.
ET AL.
single strong precipitin line was formed between the antiserum and purified Tilapia GH. A line of partial identity to the Tilapia GH formed with a pituitary extract from another teleost, the perch (Banerodon furcatus). No reaction was observed with the pituitary extract of a more primitive bony fish, the sturgeon (Acipcmer trunsmontanus). A very faint precipitin line was observed with Tilapia PRL. Absorption of the antiserum with Tilapia PRL resulted in the disappearance of this line, and the absorbed antiserum was used for the radioimmunoassay studies. No precipitin line was seen with Tilupia serum, and the antiserum was not absorbed with serum. The Tilapia GH antiserum was used in a homologous radioimmunoassay system. Tilapia GH was iodinated with 125I to serve as the tracer and was also employed as standard at a dose range of 0.25 to 4 ng. Several hormones were tested in this assay system, including purified Tilapia PRL (Farmer et al., 1975, 1976b), purified amphibian, reptilian (Farmer et al., 1976a), and mammalian GHs, as well as pituitary extracts of the perch and sturgeon. The results of this preliminary investigation are presented in Fig. 5. The Tilapia alkaline extract (E) and an intermediate fraction obtained during the Tifapia GH purification (Amberlite CG-SOC) gave inhibition slopes that were parallel to but less potent than that of the purified Tilapia GH. Tilupia PRL showed a parallel inhibition slope, but its relative immunoreactivity amounted to only 0.05% of the Tilapia GH standard, which could most likely be explained by a trace contamination with GH. A significant cross-reaction was observed with pituitary extract from another teleost, the perch, although its slope was not parallel to the standard. This same extract showed a line of partial identity in agar diffusion (Fig. 4). Sturgeon pituitary extract and other purified GHs (bullfrog, snapping turtle, and rat) showed very low degrees of crossreaction as indicated by their relatively flat
TELEOST 50
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r
III1
0
3
1 2 4
I
I
I
I
16
64
250
looo
I
4Km
I
15.m
NANOGRAMS OF GROWTH HORMONE OR PITUITARY EXTRACT (PE)
FIG. 5. Radioimmunoassay with rabbit antiserum to Tilapiu GH demonstrating the relative immunoreactivity of GH in an alkaline extract (E) and in a fraction of intermediate purity (CC-SOC) from Tilapia pituitary fractionation; and the cross-reaction shown by purified GHs from mammalian, reptilian, and amphibian species and by GH in crude pituitary extracts of a teleost (perch) and a primitive bony fish (sturgeon). The cross-reaction with Tilupiu PRL indicates that this material has a 0.05% contamination with GH. The antiserum was absorbed with Tihpiu PRL and used at a final dilution of 1:30,000. Tilupiu GH was iodinated with iz51 according to the method of Greenwood et al. (1963) and was also used for the standard. All points represent the means of two determinations.
slopes and did not give any detectable cipitin lines in agar.
pre-
DISCUSSION
This is the first report in recent years of the preparation and characterization of a highly purified teleost GH. The purified Tilapia GH was found to be capable of stimulating growth of the tibia1 cartilage plate of rats when used at high doses, in contrast to the negative results previously obtained with crude pituitary extracts of
HORMONE
97
several species of teleost fish (Solomon and Greep, 1959; Moudgal and Li, 1961; Hayashida, 1970). The Tilapia GH also demonstrated a low but significant degree of immunochemical relatedness to mammalian GH and snapping turtle GH (Fig. 3) which had not been observed previously with teleost pituitary extracts (Hayashida, 1970; Hayashida et al., 1975). These data, and the chemical characterization of the Tilapia GH, provide strong evidence for the conservation of the GH molecular structure during evolution. The Tilapia GH behaved identically to mammalian, avian, reptilian, and amphibian GHs in the purification procedures employed (Farmer et al., 1974, 1976a). The behavior of the Tilapia GH on Sephadex G-100, in the ultracentrifuge, and on disc gel electrophoresis all indicate a marked similarity to tetrapod GHs. The elution pattern of the Tilapia GH in exclusion chromatography gives evidence for a homogeneous preparation with a Stokes radius and molecular weight very close to those of human GH monomer (Bewley, unpublished results). Although the three amino-terminal residues of Tilapia GH do not include phenylalanine, which has been found at the first or second position of the NH, terminus of all of the tetrapod species examined to date (Wilhelmi, 1955; Farmer et al,, 1974, 1976a), they are all single base mutations of phenylalanine. The presence of proline in the next position is homologous with observations on sheep, duck, pigeon, snapping turtle, and bullfrog GHs. The amino acid composition of all the tetrapod GHs studied to date have demonstrated certain features which can be said to characterize GHs (detailed above). The fact that the Tilapia GH was found to possess all of these characteristic features lends additional support to the hypothesis of conservation of structure in GH. However, as might be expected from the low biological activity of Tilapia GH in the rat and the low immunological reactivity observed with
98
FARMER
ET AL.
antisera to rat GH or turtle GH, comparison phenylalanine bands in Tilapia GH results of the amino acid composition of Tilapia from the somewhat lower content of this GH with that of both mammalian GH (Ta- amino acid in the fish protein in comparison ble 1) and other tetrapod GHs (Farmer et with the mammalian GHs. al., 1976a) reveals some notable differTwo other reports on the purification of ences. The Tilupia GH is unique from all fish GH have appeared in the literature other species in its high serine and low (Wilhelmi, 1955; Lewis et al., 1972). It is methionine and phenylalanine contents. It difficult to make direct comparisons of their has a lower amount of alanine, similar to findings with those of the present study bethe frog GHs, than is found in the sheep, cause *the purification and characterization duck, or turtle GHs. However, in most procedures were quite different. In addicases in which there are significant differtion, the vast multitude of existing fish ences in amino acid content, this fish GH is species demands the awareness of possible species differences when comparing data more closely related to the GHs of birds from any one species with those from and reptiles, and even to mammalian GH, than to the frog GHs. another species. Despite these limitations, The CD spectrum of Tilapiu GH shows a few observations can be made. Wilhelmi clearly that, despite the great evolutionary (1955) noted that hake and pollack GH also distance between it and the mammalian fractionated identically to several mammaGHs, all these molecules are characterized lian GHs, which he prepared concurrently. by an a-helix content of about 50% (Bewley However, these fish GHs were inactive in the tibia assay at a total dose of 8 mg, et al., 1969; Bewley and Li, 1972b; Holladay et al., 1974). The positive band at 299 whereas Tilapia GH gave a significant renm can be assigned to tryptophan and is sponse with 1.2 mg. This may relate to similar to the positive indole bands seen in species differences or to the fact that the Tifupiu GH was more highly purified. the CD spectra of human GH and ovine prolactin (Bewley et al., 1969; Bewley and Lewis et al. (1972) isolated a ‘ ‘GH-like proLi, 1972a). The negative band at 292 nm can tein” from shark pituitaries which was similar to the Tilapiu GH in demonstrating a also be assigned to tryptophan and is similar to negative indole bands seen at this stained band in disc gel electrophoresis as well as in its molecular weight (22,000 dalwavelength in human chorionic somatotons). Lewis observed several disc elecmammotropin (HCS) (Bewley and Li, 1971), and both bovine and ovine GHs trophoretic bands in his preparation after purification, similar to our results (Fig. 3), (Bewley and Li, 1972b; Holladay et al., 1974). The negative band at 285 nm and the ‘but only a single band with fresh pituitary shoulder around 280 nm are mostly due to extracts. He concluded that the appearance tyrosine residues, although tryptophan and of the additional bands represents deamidathe disulfide bonds may also contribute in tion occurring during purification. this region of the spectrum (Bewley and Li, The rabbit antiserum to Tilupia GH was 1971, 1972b; Holladay et a/., 1974). These found to give good precipitin lines in agar diffusion and to be suitable for developbands are somewhat similar to tyrosine Preliminary bands seen in HCS (Bewley and Li, 1971) ment of a radioimmunoassay. data were obtained and rat GH (Holladay et al., 1974). The radioimmunoassay which demonstrated the ability of the anweak bands at 269 nm and the shoulder between 260 and 265 nm can be assigned to tiserum to react with Tilapia GH in crude pituitary extracts. The assay has not yet phenylalanine residues (Bewley and Li, 1972b; Holladay et al., 1974). Perhaps the been employed for the measurement of cirof the culating GH levels in Tilapia, nor has it relatively weak appearance
TELEOST
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been utilized for studying immunochemical relatedness of GH from representatives of various orders of fishes. These studies are just commencing and hopefully may lead to a better insight into both the physiology of GH in teleost fishes as well as phylogenetic relationships among this vast group of fishes. It is of interest that of the pituitary extracts and purified GHs from other species that were tested, only that of another teleost showed a significant degree of cross-reaction. These results are in contrast to the extensive species crossreactivity observed with antiserum to rat and snapping turtle GHs (Hayashida, 1970; Hayashida et al., 1975). ACKNOWLEDGMENTS The collection of Tilapia pituitaries was made possible by a grant to H. A. B. from the Graduate Division of the University of California, Berkeley, and was accomplished by a team under the leadership of R. S. Nishioka, consisting of Lauren Bern, Peggy Broadley, Karen T. Mills, Daniel Swanson, and L. Jay Wiley. To them, to the students of the Aiea High School Biology Club and their advisor, Ms. Iris Shinseki, and Mr. William S. Devick of the Hawaiian Department of Land and Natural Resources, Division of Fish and Game, we express our appreciation. We thank Jean Knorr, Kenway Hoey, .I. D. Nelson, Eleanor Rowley, William Lindsey, and Donald Bruschera for expert technical assistance. These studies were supported in part by grants from the National Science Foundation (BMS 75-16138 and GB-35239) and the National Institutes of Health (HD-04063).
REFERENCES Beaven, G. H., and Holiday, E. R. (1952). Ultraviolet absorption spectra of proteins and amino acids. Advan. Protein Chem. 7, 319-386. Bewley, T. A., and Li, C. H. (1971). Circular dichroism studies on human chorionic somatomammotropin. Arch. Biochem. Biophys. 144, 589-595. Bewley, T. A., and Li, C. H. (1972a). Circular dichroism studies on human pituitary growth hormone and ovine pituitary lactogenic hormone. Biochemistry 11, 884-888. Bewley, T. A., and Li, C. H. (1972b). Molecular weight and circular dichroism studies of bovine and ovine pituitary growth hormone. Biochemistry 11, 927-931.
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Bewley, T. A., Brovetto-Cruz, J., and Li, C. H. (1969). Human pituitary growth hormone. Physicochemical investigations of the native and reduced-alkylated protein. Biochemistry 8, 4701-4708. Bewley, T. A., Kawauchi, H., and Li, C. H. (1972). Comparative studies of the single tryptophan residue in human chorionic somatomammotropin and human pituitary growth hormone. Biochemistry 11, 4179-4187. Cheever, E. V., and Lewis, U. J. (1969). Estimation of the molecular weights of the multiple components of growth hormone and prolactin. Endocrinology 85, 465-473. Clarke, W. C. (1973). Sodium-retaining bioassay of prolactin in the intact teleost Tilupia mossambica acclimated to sea water. Gen. Comp. Endocrinol. 21,
498-513.
Doneen, B. A. and Bern, H. A. (1974). In vitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillichthys mirabilis. J. Exp. Zool. 187, 173-179. Edman, P. (1950). Method for determination of the amino acid sequence in peptides. Acta Chem. &and. 4, 283-293. Ellis, S., Grindeland, R. E., Nuenke, J. M., and Callahan, P. X. (1968). Isolation and properties of rat and rabbit growth hormones. Ann. N.Y. Acad. Sci. 148, 328-342. Farmer, S. W., Clarke, W. C., Papkoff, H., Nishioka, R. S., Bern, H. A., and Li, C. H. (1975). Studies on the purification and properties of teleost prolactin. Life Sci. 16, 149-159. Farmer, S. W., Papkoff, H., and Hayashida, T. (1974). Purification and properties of avian growth hormones. Endocrinology 95, 1560-1565. Farmer, S. W., Papkoff, H., and Hayashida, T. (1976a). Purification and properties of reptilian and amphibian growth hormones. Endocrinology, 99, in press. Farmer, S. W., Papkoff, H., Bewley, T. A., Hayashida, T., Nishioka, R. S., Bern, H. A., and Li, C. H. (1976b). Purification and properties of teleost prolactin. Gen. Comp. Endocrinol., submitted for publication. Gray, W. R. (1967). Sequential degradation plus dansylation. Methods Enzymol. 11, 469-475. 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. Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963). The preparation of ‘3’I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. Hayashida, T. (1970). Immunological studies with rat pituitary growth hormone (RGH). II. Compara-
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FARMER
tive immunochemical investigation of GH from representatives of various vertebrate classes with monkey antiserum to RGH. Gen. Camp. 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 extracts of elasmobranchs. Gen. Camp. Endocrinol. 20, 377-385. Hayashida, T., and Contopoulos, A. N. (1967). Immunological studies with rat pituitary growth hormone. I. Basic studies with immunodiffusion and antihormone tests. Gen. Camp. Endocrinot. 9, 217-226. Hayashida, T., Farmer, S. W., and Papkoff, H. (1975). Pituitary growth hormones: Further evidence for conservatism based on imevolutionary munochemical studies. Proc. Nat. Acad. Sci. USA 72, 4322-4326. Hayashida, T., and Lagios, M. (1969). Fish growth hormone: A biological, immunochemical and ultrastructural study of sturgeon and paddlefish pituitaries. Gen. Comp. Endocrinol. 13,407-411. Holladay, L. A., Hammonds, R. G. Jr., and Puett, D. (1974). Growth hormone conformation and conformational equilibria. Biochemistry 13, 16531661. Laurent, T. C., and Killander, J. (1964). A theory of gel filtration and its experimental verification. J. Chromatogr. 14, 317-330. Lewis, U. J., Singh, R. N. P., Seavey, B. K., Lasker, R., and Pickford, G. E. (1972). Growth hormoneand prolactin-like proteins of the blue shark (Prionace glauca). Fish. Bull. 70, 933-939. Lorenson, M. G., and Ellis, S. (1975). Amino terminal residue heterogeneity of bovine and ovine growth hormones as revealed by polyacrylamide gel electrophoresis. Endocrinology 96, 833-838. Li, C. H. (1954). A simplified procedure for the isolation of hypophyseal growth hormone. J. Biol. Chem. 211, 555-558. Li, C. H. (1957). Studies on pituitary lactogenic hormone. XVII. Oxidation of the ovine hormone with performic acid. J. Biol. Chem. 229, 157-163. Li, C. H., Gordon, D., and Knorr, J. (1973). The primary structure of sheep pituitary growth hormone. Arch. Biochem. Biophys. 156, 493-508. Moore, S. (1972). In “Chemistry and Biology of Peptides” (J. Meienhofer, ed.), p. 629. Ann Arbor Science, Ann Arbor, Mich.
ET AL. Moudgal, N. R., and Li, C. H. (1961). Immunochemical studies of bovine and ovine pituitary growth hormone. Arch. Biochem. Biophys. 93, 122-127. Niu, C. I., and Fraenkel-Conrat, H. (1955). Determination of C-terminal amino acids and peptides by hydrazinolysis. J. Amer. Chem. Sot. 72, 58825885. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Ann. N. Y. Acad. Sci. 121, 321-349. Ouchterlony, 0. (1953). Antigen-antibody reactions in gel; types of reactions in coordinated systems of diffusion. Acta Pathol. Microbial. Stand. 32, 231-240. Papkoff, H., and Li, C. H. (1958). The isolation and characterization of growth hormone from anterior lobes of whale pituitaries. J. Biol. Chem. 231, 367-377. Papkoff, H., Li, C. H., and Liu, W-K. (1962). The isolation and characterization of growth hormone from porcine pituitaries. Arch. Biochem. Biophys. 96, 216-225. Pickford, G. E. (1954). The response of hypophysectomized male killifish to purified fish growth hormone, as compared with the response to purified beef growth hormone. Endocrinology 55, 274287. Pickford, G. E., and Atz, J. W. (1957). In “The Physiology of the Pituitary Gland of Fishes”, p. 91, New York Zoological Society, New York. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1962). Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature (London) 195, 281-283. Solomon, J., and Greep, R. 0. (1959). The growth hormone content of several vertebrate pituitaries. Endocrinology 65, 334-335. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. Wilhelmi, A. E. (1955). In “The Hypophyseal Growth Hormone, Nature and Actions” (R. W. Smith, Jr., 0. H. Gaebler, and C. N. H.. Long, eds.), p. 69. McGraw-Hill (Blakiston), New York. Wilhelmi, A. E. (1974). In “Handbook of Physiology” (E. Knobil and W. H. Sawyer, eds.), Sect. 7, Vol. IV, Part 2, p. 59. American Physiological Society, Washington, DC. Woods, K. R., and Wang, K. T. (1967). Separation of dansyl-amino acids by polyamide layer chromatography. Biochim. Biophys. Acta 133, 369-370.
AND
COMPARATIVE
ENDOCRINOLOGY
Purification
30,
91-100 (1976)
and Properties
of Teleost Growth Hormone
SUSAN WALKER FARMER, HAROLD PAPKOFF, TED HAYASHIDA,’ THOMAS A. BEWLEY,HOWARD A. BERN,~ AND CHOH HAO LI The Hormone 2Department
Research
Laboratory and ‘Department of Anatomy, University of California, San Francisco, California 94143 and of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720
Accepted May 7, 1976 Highly purified growth hormone (GH) has been prepared from the pituitary glands of a euryhaline teleost, Tilapia mosambica. Tilapia GH was obtained in a yield of 1400 mg/kg wet weight tissue. It was found to have a molecular weight (gel filtration) of 22,200 daltons, a sedtmentatton coeffictent (s~~,~) of 2.19, and an a-helix content (circular dichroism) of 50%. Isoleucine was found to be the major amino-terminal residue; leucine was found to be COOH terminal. The amino acid composition, disc gel electrophoresis pattern, and circular dichroism spectra were similar to those of mammalian GHs. Tilapia GH was found to have a low but significant activity in the rat tibia assay and showed immunological relatedness to mammalian GH in a rat GH radioimmunoassay. Antiserum was prepared against the Tilapia GH and characterized in agar diffusion experiments and radioimmunoassay. Results from these investigations demonstrated a significant degree of cross-reaction between Tilapia GH and pituitary extract from another teleost (perch), but purified tetrapod GHs were essentially nonreactive. The data indicate a significant resemblance between Tilapia GH and mammalian GHs and suggest that the GH structure has been strongly conserved during evolution.
The presence of a growth hormone (GH) in fish pituitaries has been clearly established. Hypophysectomy of fish causes a cessation of growth, and replacement therapy with either fish pituitary extracts or mammalian GH causes a resumption of growth (Pickford and Atz, 1957). However, attempts by several workers to test pituitary extracts of modem bony fish (teleost) in the rat tibia assay have given negative results (Solomon and Greep, 1959; Moudgal and Li, 1961; Hayashida, 1970). Interestingly, pituitary extracts of primitive bony fish (Hayashida and Lagios, 1969), holosteans (Hayashida, 1971), and elasmobranchs (Hayashida, 1973), were shown to be active in the rat tibia assay. Similarly, when pituitary extracts of these fish were tested for cross-reaction in a rat GH radioimmunoassay, those of teleosts showed no appreciable immunochemical relatedness to
mammalian GH, while those of primitive bony fishes, holosteans, and elasmobranchs showed low but significant cross-reaction with the rat GH antiserum (Hayashida, 1970). These findings, coupled with the recently available comparative information on purified GHs from other nonmammalian species (avian, reptilian, and amphibian; Farmer et al., 1974,1976a), make a study of purified fish GH of special interest. However, attempts to purify fish GH have been severely hindered by the lack of a sensitive and convenient GH assay in fish, the inactivity of crude teleost pituitary extracts in the tibia assay, and the difficulty in obtaining sufficient quantities of fish pituitaries. Wilhelmi (1955) described two preparations of GH, from hake and pollack pituitaries, which stimulated growth in fish (Pickford, 1954) but were inactive in the rat tibia as91
Copyright @ 1976 by Academic Press. Inc. AI! rights of reproduction in any form reserved.
92
FARMER
say. Lewis et al. (1972), using disc electrophoresis for identification, purified a “GH-like protein” from shark pituitaries which was also found to stimulate growth in teleost fish. This report presents data on the isolation and characterization of highly purified GH teleost, Tilapia from a euryhaline mossambica.3 During our isolation of prolactin (PRL) from this species (Farmer et al., 1975, 1976b) all fractions were examined by disc gel electrophoresis. A side fraction from this purification was found to give a pattern that has been characteristic for mammalian GHs (Cheever and Lewis, 1969). This fraction was purified and chemically characterized. Activity in the rat tibia assay confirmed the identity of this material as teleost GH. MATERIALS
AND METHODS
Pituitaries. Mature Tilapia mossambica of both sexes were collected in Wahiawa Reservoir, Hawaii; the pituitaries were removed immediately, frozen, and kept at -20” until fractionation. A total of 20,001 pituitaries were obtained which amounted to 63 g wet weight. Fractionation. Fractionation methodology was similar to that used in our laboratory for the purification of GH from mammalian and nonmammalian species (Papkoff and Li, 1958; Papkoff et al., 1%2; Farmer et al., 1974, 1976a). The initial purification steps were also identical to those reported for the isolation of Tilapia PRL (Farmer et al., 1975) up to the point where PRL and GH were separated. In brief, the pituitaries were homogenized in a Waring Blendor with cold distilled water, adjusted to pH 9.5 with Ca(OH),, and stirred at 4” for 3 hr. After centrifugation, the alkaline extract was dialyzed, lyophilized, and then dissolved in phosphate buffer (pH 5.1), containing 12% saturated (NH&SO, for chromatography on Amberlite CG-50 as previously described (Papkoff et al., 1962). The fraction eluting with pH 6.0 buffer was shown to contain both the PRL and GH. Subsequent chromatography on DEAEcellulose, equilibrated with 0.03 M NH.,HCO,, pH 9, effected a separation of the PRL and GH; PRL was unadsorbed (Farmer et al., 1975) and GH was adsorbed and eluted with 0.2 M NKHCO,.
3 This Sarotherodon
species
has
mossambicus.
recently
been
renamed
ET AL.
Further purification of Tilapia GH was obtained by a series of precipitations at various pH values and salt and alcohol concentrations: Tilapia GH precipitated at pH 5.0; at pH 5.3 in the presence of 20% ethanol; at pH 3.5 adjusted with HPO, in the presence of 0.15 M (NH&SO,; and in 40% saturated (NH&SO,. In each case impurities remained in the supematant. Final purification was obtained by gel filtration on Sephadex G-100 equilibrated with 0.05 M NH4HC03. The material eluting with a VJVO of 2.1 was used for characterization after refiltration. Chemical and physical characterization. Pertinent fractions were examined by disc gel electrophoresis at pH 4.5 (Reisfeld et a/., 1962) and pH 8.3 (Omstein, 1964) in 7.5% acrylamide gels stained with amido schwarz. NH,-terminal analyses were performed by the dansyl technique (Gray, 1967; Woods and Wang, 1967); NH,-terminal sequence by the dansyl-Edman procedure (Gray, 1967; Woods and Wang, 1%7; Edman, 1950); COOH-terminal analyses by the carboxypeptidase reaction as previously described (Farmer et al., 1976a) and by hydrazinolysis (Niu and Fraenkel-Conrat, 1955); and amino acid analyses by the method of Spackman et al. (1958) in an automatic amino acid analyzer (Beckman Model 120B). Sedimentation studies on the Tilapia GH were performed in a Spinco Model E ultracentrifuge at a speed of 59,780 rpm, at room temperature. The hormone was dissolved in 0.1 M Tris-HCI, pH 8.2, at a concentration of 1 mg/ml. The molecular weight of Tilapia GH was estimated by exclusion chromatography on a column of Sephadex G-100 (1.5 x 59 cm) in 0.1 M Tris-HC1 buffer (pH 8.2). The sample was applied to the bottom of the column and eluted upward at a flow rate of 4.9 ml/hr using an LKB Model 10200 peristaltic pump. Elution patterns were continuously monitored near 280 nm with an LKB Uvicord-II. The column was calibrated with blue dextran 2000 (Pharmacia), myoglobin, bovine serum albumin (Miles Research Laboratories), and highly purified human GH. Stokes radii were calculated from the elution patterns as described by Laurent and Killander (1964). Circular dichroism (CD) spectra were recorded on a Cary Model 60 spectropolarimeter equipped with a Model 6002 circular dichroism attachment according to procedures previously outlined (Bewley et al., 1972). The content of a-helix was estimated as described elsewhere (Bewley et at., 1969). Protein concentrations were determined from absorption spectra taken in the Tris-HCl buffer on a Beckman DK-2A spectrophotometer. Spectra were recorded from 360 to 245 nm and corrected for light scattering as described by Beaven and Holiday (1952). An absorptivity value, E, c,,,,*,, n,“.l% = 0.975, was determined from the spectrum of a carefully weighed sample of Tilapia GH dissolved in the Tris-HCl buffer (pH 8.2),
TELEOST
GROWTH
after correcting for light scattering and assuming a 15% moisture content for the lyophilized protein. Bioassay. The Tilapia GH was tested for biological activity in the rat tibia assay (Greenspan ef al., 1949). Female Long-Evans rats were hypophysectomized at 28 days of age and used I days postoperatively for the assay. Highly purified bovine GH prepared in this laboratory (Li, 1954) was used as a standard. Immunochemistry. The immunochemical reactivity of Tilupia GH was tested with rat GH antiserum (Hayashida and Contopoulos, 1967) and snapping turtle GH antiserum (Hayashida et al., 1975), in double antibody radioimmunoassay systems. The techniques employed in the radioimmunoassays have been previously described (Hayashida, 1971). Purified rat GH (3.0 USP Ulmg; Ellis et al., 1968) was labeled with lz51 (Greenwood et al., 1963) and was also employed as standard. The second antibody used for the immunoprecipitation of the labeled rat GH was rabbit anti-human y-globulin serum. In addition, antiserum was produced against the Tilapia GH. A single young albino rabbit was immunized with a total of 4.5 mg of Tilupia GH. Four injections of 1 mg each were given in complete Freund’s adjuvant at weekly intervals. Each injection was divided into three equal portions, two being given SCand one ip. A booster injection of 0.5 mg was given in saline 10 days after the last weekly injection. The animal was bled 1 week after the booster injection and tested for the presence of precipitating antibodies by the Ouchterlony agar diffusion technique (Ouchterlony, 1953). The results were positive, and the animal was bled completely and sacrificed. Merthiolate (0.01%) was added to the serum as a preservative. The agar diffusion test with the antiserum revealed no precipitin line with Tilapia serum but a faint line with Tilapia PRL. Therefore the antiserum was absorbed with Tilapia PRL, as described by Hayashida (1970). After absorption, no precipitin line was observed with Tilapia PRL. The absorbed antiserum was used at a final dilution of 1:30,000 in a double antibody radioimmunoassay system, similar to that previously described (Hayashida, 1971). Tilapia GH was iodinated with rZsI by the method of Greenwood ef al. (1963) and was also employed as the standard.
RESULTS Chemical and Physical Characterization The Tilapia GH behaved identically to
known mammalian and nonmammalian GHs during fractionation, and a yield of 90 mg was obtained, which represents 1400 mg/kg wet weight tissue. This yield is comparable to the value of 2000 mg/kg obtained
HORMONE
93
for bovine GH (Li, 1954). The final purification step was gel filtration on Sephadex G-100, and the Tilapia GH emerged as a single symmetrical peak with a VJV,, of 2.1. This GH also showed a single symmetrical sedimenting boundary in the ultracentrifuge and a sedimentation coefficient (sz,& of 2.19 was calculated. Exclusion chromatography on a calibrated Sephadex G-100 column was performed to determine the molecular weight of the Tilapia GH. The GH exhibited a single, symmetrical peak with a V$V, ratio of 2.09. A Stokes radius of 22.9 A and a molecular weight of 22,200 daltons were calculated for Tilapia GH from its elution characteristics. These values may be compared with a VJV, of 2.10, a Stokes radius of 22.6 A, and a molecular weight of 22,100 daltons obtained for human GH monomer on this column (Bewley, unpublished results). During the purification of the Tilapia GH, the presumptive GH fraction was identified by polyacrylamide disc gel electrophoresis. The pattern obtained with the purified Tilapia GH at pH 8.3, shown in Fig. 1, has a major heavily stained band and two faster-moving, less intensely staining bands. An identical pattern has been found with representative species in each class of vertebrates (Farmer et al., 1976a). The two faster-moving bands may be deamidated forms of GH (Cheever and Lewis, 1969) or a reflection of microheterogeneity at the NH,-terminal residue (Lorenson and Ellis, 1975). The latter possibility was demonstrated to be true in the case of the Tilapia GH (see below). The fact that these additional bands are not indicative of the presence of other proteins is supported by the finding of a single band with this GH at pH 4.5 (Fig. 1). The observation that stained bands were not seen in other regions of the gel also indicates the absence of contaminating proteins. NH,-terminal analysis by the dansyl procedure showed the presence of isoleucine,
94
FARMER
ET AL.
TABLE AMINO
Amino acid LYS His Arg Asp Thr Ser Glu pro
GUY Ala ‘h-cys Val Met Ile Leu W Phe Trp
FIG. 1. Disc electrophoretic pattern of Tilapia GH at pH 4.5 (left) and pH 8.3 (right), 50 pg each on 7.5% gels stained with amido schwarz.
leucine, and valine in approximate amounts of 70, 20, and lo%, respectively. However, application of the dansyl-Edman procedure showed only a single residue, proline, at the next position. At the carboxyl terminal, a single residue, leucine, was found both by the hydrazinolysis reaction and by liberation with carboxypeptidase. These results suggest that the variation seen at the NH, terminus may represent single base mutations of the same basic molecule. The amino acid analysis of Tilapia GH is shown in Table 1, with that of ovine GH included for comparison. Because Tilapia GH was found to have a molecular weight similar to that of ovine GH, the amino acid analysis was calculated on the basis of Tilapia GH having the same number of res-
1
ACID COMPOSITION OF Tilapia GROWTH HORMONES COMPARED WITH OVINE GROWTH HORMONES
Ovine growth hormone 11 3 13 16 12 13 24 6 10 15 4 6 4 7 27 6 13 1
Tilapia
growth hormone 8.2 5.0 11.0 19.3 12.0 21.4 29.1 6.8 7.4 8.2 4.6 6.0 1.2 9.0 21.2 7.2 6.7 1.0
a Amino acid analysis: 20-hr hydrolysis, not corrected for hydrolytic destruction, calculated on the basis of 191 residues/mole; cystine and methionine values were obtained with performic acid-oxidized preparations (Li, 1957); the tryptophan value was determined by methane sulfonic acid hydrolysis (Moore, 1972) and by spectrophotometric measurement (Beaven and Holiday, 1952). All values are residues per mole. JJTaken from structural analysis of ovine GH, 191 residues (Li et al., 1973).
idues as ovine GH: 191 (Li et al., 1973). The Tilapia GH exhibits the features that have been found to characterize mammalian GHs, namely, two disulfides, a single tryptophan, low methionine and histidine content, and a high glutamic acid and leucine content (Wilhelmi, 1975). However, there are also notable differences between the two species: Tilapia GH has a high aspartic acid and serine content and a low methionine, and phenylalanine alanine, content compared with ovine GH. The circular dichroism spectra obtained with Tilapia GH are shown in Fig. 2. In the
TELEOST
GROWTH t12.5 -i
95
HORMONE T
B
0
d -‘2.5 1 -25.0 p -37.5 “5 -50.0 3
-625
-
-75.0 260
270
280
6 290
300
310
WAVELENGTH (NM)
FIG. 2. Circular dichroism spectra of Tikzpia GH in 0.1 M Tris-HCI buffer (pH 8.2), in the region of amide bond absorption (A) and in the region of side-chain absorption (B).
region dominated by amide bond absorption, the CD spectrum of Tilupia GH shows the two negative bands at 209 and 221 nm which are characteristic of the o-helix. To the nearest 5%, an a-helix content of 50% may be estimated for Tilupia GH from the ellipticities at either 209 or 221 nm. In the region of side-chain absorption, the CD spectrum of Tilapia GH exhibits a weak, positive band near 299 nm, two stronger, negative bands at 292 and 285 nm, a negative shoulder around 280 nm, a weak negative band at 269 nm, and a weak negative shoulder between 260 and 265 nm. Biological Characterization
The purified Tilapia GH was tested in the commonly employed mammalian growth hormone bioassay, the tibia test (Greenspan et al., 1949). It gave a low but significant response which was dose related, although not parallel to the bovine GH response (Table 2). The Tilupia GH TABLE Assay
2
OF BOVINE AND Tilapia BY THE TIBIA TEST
Preparation
Dose OLP)
Number of animals
Saline Bovine GH Bovine GH Tilapia GH Tilupia GH
20 60 400 1200
4 4 4 4 4
GH
Tibia width (wm k SEM) 181 k 199 !I 258 f 200 k 220 2
4 3 6 9 8
was also tested in two fish PRL bioassays. In the sodium-retention assay in Tilupia (Clarke, 1973), the Tilapia GH was inactive at a dose of 10 pg, and it was inactive at a dose of 50 pg in the Gillichthys urinary bladder assay (Doneen and Bern, 1974). Tilapia PRL was active in the same assays at doses of 0.1 and 1 pg, respectively (Farmer et al., 1976b). Immunochemical Characterization
The Tilapia GH was tested for immunochemical reactivity in two radioimmunoassay systems, employing rat GH antiserum (Hayashida, 1970) and snapping turtle GH antiserum (Hayashida et al., 1975). Pituitary extracts of teleost fish previously tested had shown extremely weak or no cross-reaction in these assays. Puritied Tilapia GH, however, significantly inhibited the binding of labeled rat GH in both assay systems (Fig. 3), although relatively large quantities of the hormone were required. The inhibition slope for Tilupia GH was parallel to that of the rat GH standard in the turtle GH antiserum system, but it was not quite parallel to the standard in the rat GH antiserum system. In preliminary studies the Tilapia GH antiserum was evaluated for specificity and cross-immunoreactivity. Shown in Fig. 4 is an Ouchterlony agar diffusion plate with Tilupia GH antiserum in the center well and various antigens in the peripheral wells. A
96
FARMER
:,
0
III/ .5 1 2 4 NANOGRAMS
I 16
I 64
I 250
I loo0
I , AC03 15,000
OF PURIFIED HORMONE
FIG. 3. Cross-reaction of purified Tilapiu GH in two different radioimmunoassays, one employing monkey antiserum to rat GH (solid lines) and the other employing monkey antiserum to snapping turtle GH (dashed lines). Highly purified rat GH (Ellis, 3.0 USP U/mg) was iodinated with 9 according to the method of Greenwood et al. (1%3) and was also used for the standard. All points are the averages of two determinations.
FIG. 4. Ouchterlony plate showing the reaction of unabsorbed rabbit serum to Tilapia GH (center well, 0.15 ml) with purified i”i~upia GH (10 pg), perch pituitary extract, sturgeon pituitary extract, rat GH (10 CLp), Tilapia PRL (20 pg), and normal Tilapiu serum (0.05 ml, 1:lO dilution). The pituitary extracts are the equivalent of 1.O mg of wet tissue weight/well.
ET AL.
single strong precipitin line was formed between the antiserum and purified Tilapia GH. A line of partial identity to the Tilapia GH formed with a pituitary extract from another teleost, the perch (Banerodon furcatus). No reaction was observed with the pituitary extract of a more primitive bony fish, the sturgeon (Acipcmer trunsmontanus). A very faint precipitin line was observed with Tilapia PRL. Absorption of the antiserum with Tilapia PRL resulted in the disappearance of this line, and the absorbed antiserum was used for the radioimmunoassay studies. No precipitin line was seen with Tilupia serum, and the antiserum was not absorbed with serum. The Tilapia GH antiserum was used in a homologous radioimmunoassay system. Tilapia GH was iodinated with 125I to serve as the tracer and was also employed as standard at a dose range of 0.25 to 4 ng. Several hormones were tested in this assay system, including purified Tilapia PRL (Farmer et al., 1975, 1976b), purified amphibian, reptilian (Farmer et al., 1976a), and mammalian GHs, as well as pituitary extracts of the perch and sturgeon. The results of this preliminary investigation are presented in Fig. 5. The Tilapia alkaline extract (E) and an intermediate fraction obtained during the Tifapia GH purification (Amberlite CG-SOC) gave inhibition slopes that were parallel to but less potent than that of the purified Tilapia GH. Tilupia PRL showed a parallel inhibition slope, but its relative immunoreactivity amounted to only 0.05% of the Tilapia GH standard, which could most likely be explained by a trace contamination with GH. A significant cross-reaction was observed with pituitary extract from another teleost, the perch, although its slope was not parallel to the standard. This same extract showed a line of partial identity in agar diffusion (Fig. 4). Sturgeon pituitary extract and other purified GHs (bullfrog, snapping turtle, and rat) showed very low degrees of crossreaction as indicated by their relatively flat
TELEOST 50
GROWTH
r
III1
0
3
1 2 4
I
I
I
I
16
64
250
looo
I
4Km
I
15.m
NANOGRAMS OF GROWTH HORMONE OR PITUITARY EXTRACT (PE)
FIG. 5. Radioimmunoassay with rabbit antiserum to Tilapiu GH demonstrating the relative immunoreactivity of GH in an alkaline extract (E) and in a fraction of intermediate purity (CC-SOC) from Tilapia pituitary fractionation; and the cross-reaction shown by purified GHs from mammalian, reptilian, and amphibian species and by GH in crude pituitary extracts of a teleost (perch) and a primitive bony fish (sturgeon). The cross-reaction with Tilupiu PRL indicates that this material has a 0.05% contamination with GH. The antiserum was absorbed with Tihpiu PRL and used at a final dilution of 1:30,000. Tilupiu GH was iodinated with iz51 according to the method of Greenwood et al. (1963) and was also used for the standard. All points represent the means of two determinations.
slopes and did not give any detectable cipitin lines in agar.
pre-
DISCUSSION
This is the first report in recent years of the preparation and characterization of a highly purified teleost GH. The purified Tilapia GH was found to be capable of stimulating growth of the tibia1 cartilage plate of rats when used at high doses, in contrast to the negative results previously obtained with crude pituitary extracts of
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several species of teleost fish (Solomon and Greep, 1959; Moudgal and Li, 1961; Hayashida, 1970). The Tilapia GH also demonstrated a low but significant degree of immunochemical relatedness to mammalian GH and snapping turtle GH (Fig. 3) which had not been observed previously with teleost pituitary extracts (Hayashida, 1970; Hayashida et al., 1975). These data, and the chemical characterization of the Tilapia GH, provide strong evidence for the conservation of the GH molecular structure during evolution. The Tilapia GH behaved identically to mammalian, avian, reptilian, and amphibian GHs in the purification procedures employed (Farmer et al., 1974, 1976a). The behavior of the Tilapia GH on Sephadex G-100, in the ultracentrifuge, and on disc gel electrophoresis all indicate a marked similarity to tetrapod GHs. The elution pattern of the Tilapia GH in exclusion chromatography gives evidence for a homogeneous preparation with a Stokes radius and molecular weight very close to those of human GH monomer (Bewley, unpublished results). Although the three amino-terminal residues of Tilapia GH do not include phenylalanine, which has been found at the first or second position of the NH, terminus of all of the tetrapod species examined to date (Wilhelmi, 1955; Farmer et al,, 1974, 1976a), they are all single base mutations of phenylalanine. The presence of proline in the next position is homologous with observations on sheep, duck, pigeon, snapping turtle, and bullfrog GHs. The amino acid composition of all the tetrapod GHs studied to date have demonstrated certain features which can be said to characterize GHs (detailed above). The fact that the Tilapia GH was found to possess all of these characteristic features lends additional support to the hypothesis of conservation of structure in GH. However, as might be expected from the low biological activity of Tilapia GH in the rat and the low immunological reactivity observed with
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antisera to rat GH or turtle GH, comparison phenylalanine bands in Tilapia GH results of the amino acid composition of Tilapia from the somewhat lower content of this GH with that of both mammalian GH (Ta- amino acid in the fish protein in comparison ble 1) and other tetrapod GHs (Farmer et with the mammalian GHs. al., 1976a) reveals some notable differTwo other reports on the purification of ences. The Tilupia GH is unique from all fish GH have appeared in the literature other species in its high serine and low (Wilhelmi, 1955; Lewis et al., 1972). It is methionine and phenylalanine contents. It difficult to make direct comparisons of their has a lower amount of alanine, similar to findings with those of the present study bethe frog GHs, than is found in the sheep, cause *the purification and characterization duck, or turtle GHs. However, in most procedures were quite different. In addicases in which there are significant differtion, the vast multitude of existing fish ences in amino acid content, this fish GH is species demands the awareness of possible species differences when comparing data more closely related to the GHs of birds from any one species with those from and reptiles, and even to mammalian GH, than to the frog GHs. another species. Despite these limitations, The CD spectrum of Tilapiu GH shows a few observations can be made. Wilhelmi clearly that, despite the great evolutionary (1955) noted that hake and pollack GH also distance between it and the mammalian fractionated identically to several mammaGHs, all these molecules are characterized lian GHs, which he prepared concurrently. by an a-helix content of about 50% (Bewley However, these fish GHs were inactive in the tibia assay at a total dose of 8 mg, et al., 1969; Bewley and Li, 1972b; Holladay et al., 1974). The positive band at 299 whereas Tilapia GH gave a significant renm can be assigned to tryptophan and is sponse with 1.2 mg. This may relate to similar to the positive indole bands seen in species differences or to the fact that the Tifupiu GH was more highly purified. the CD spectra of human GH and ovine prolactin (Bewley et al., 1969; Bewley and Lewis et al. (1972) isolated a ‘ ‘GH-like proLi, 1972a). The negative band at 292 nm can tein” from shark pituitaries which was similar to the Tilapiu GH in demonstrating a also be assigned to tryptophan and is similar to negative indole bands seen at this stained band in disc gel electrophoresis as well as in its molecular weight (22,000 dalwavelength in human chorionic somatotons). Lewis observed several disc elecmammotropin (HCS) (Bewley and Li, 1971), and both bovine and ovine GHs trophoretic bands in his preparation after purification, similar to our results (Fig. 3), (Bewley and Li, 1972b; Holladay et al., 1974). The negative band at 285 nm and the ‘but only a single band with fresh pituitary shoulder around 280 nm are mostly due to extracts. He concluded that the appearance tyrosine residues, although tryptophan and of the additional bands represents deamidathe disulfide bonds may also contribute in tion occurring during purification. this region of the spectrum (Bewley and Li, The rabbit antiserum to Tilupia GH was 1971, 1972b; Holladay et a/., 1974). These found to give good precipitin lines in agar diffusion and to be suitable for developbands are somewhat similar to tyrosine Preliminary bands seen in HCS (Bewley and Li, 1971) ment of a radioimmunoassay. data were obtained and rat GH (Holladay et al., 1974). The radioimmunoassay which demonstrated the ability of the anweak bands at 269 nm and the shoulder between 260 and 265 nm can be assigned to tiserum to react with Tilapia GH in crude pituitary extracts. The assay has not yet phenylalanine residues (Bewley and Li, 1972b; Holladay et al., 1974). Perhaps the been employed for the measurement of cirof the culating GH levels in Tilapia, nor has it relatively weak appearance
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been utilized for studying immunochemical relatedness of GH from representatives of various orders of fishes. These studies are just commencing and hopefully may lead to a better insight into both the physiology of GH in teleost fishes as well as phylogenetic relationships among this vast group of fishes. It is of interest that of the pituitary extracts and purified GHs from other species that were tested, only that of another teleost showed a significant degree of cross-reaction. These results are in contrast to the extensive species crossreactivity observed with antiserum to rat and snapping turtle GHs (Hayashida, 1970; Hayashida et al., 1975). ACKNOWLEDGMENTS The collection of Tilapia pituitaries was made possible by a grant to H. A. B. from the Graduate Division of the University of California, Berkeley, and was accomplished by a team under the leadership of R. S. Nishioka, consisting of Lauren Bern, Peggy Broadley, Karen T. Mills, Daniel Swanson, and L. Jay Wiley. To them, to the students of the Aiea High School Biology Club and their advisor, Ms. Iris Shinseki, and Mr. William S. Devick of the Hawaiian Department of Land and Natural Resources, Division of Fish and Game, we express our appreciation. We thank Jean Knorr, Kenway Hoey, .I. D. Nelson, Eleanor Rowley, William Lindsey, and Donald Bruschera for expert technical assistance. These studies were supported in part by grants from the National Science Foundation (BMS 75-16138 and GB-35239) and the National Institutes of Health (HD-04063).
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