- Email: [email protected]
Isolation and properties of chum salmon prolactin
GENERAL
AND
COMPARATIVE
Isolation HIROSHI
School
49,
4466458 (1983)
and Properties
of Chum
ENDOCRINOLOGY
KAWAUCHI, KEN-ICHI ABE, AKIYOSHI SANAE HASEGAWA,” NOBUKO NAITO,?
of Fisheries University
Sciences, of Tokyo,
Kitasato Nakano,
Salmon
Prolactin
TAKAHASHI, TETSUYA HIFUNO,* AND YASUMITSU NAKAI?
University, Sanriku, Iwate 022-01; Tokyo 164, and tSchoo1 of Medicine Shinagawa, Tokyo 142. Japan
*Ocean Shown
Research University,
Institute,
Accepted April 19, 1982 A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mgig of wet tissue. It was lo- 1.5times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with VJV, = 2.0. Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an o-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH,-terminal residue, isoleucine, and a single COOH-terminal residue, halfcystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH,-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.
It has been suggested that prolactin (PRL) and growth hormone (GH) have evolved from a common ancestral molecule, based mainly on structural similarities between the mammalian hormones (Bewley and Li, 1971). PRLs are known to exhibit multiple actions among vertebrates, whereas GH is exclusively concerned with stimulation of animal growth (Bern, 197.5; Clarke and Bern, 1980). To obtain better insight into the evolutionary history of these hormones and the great diversity of their functions, it is essential to isolate and characterize PRLs and GHs at many phylogenetic levels. When compared with our knowledge of the chemistry of mammalian PRLs, little is known about the nonmammalian hormone. Earlier studies to isolate fish PRL had limited success because of the absence of a convenient bioassay (Clarke and Bern, 446 00 16-6480/83/030446- 13$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
1980). Tilapia PRL has now been isolated using a sodium-retaining assay and extensively characterized physicochemically (Farmer et nl., 1977). Recently, isolation and partial characterization of fish PRLs have been reported using salmon (Idler et al., 1978), carp and flounder (Ng et al., 1980). During the course of our studies on purification and characterization of the proopiocortin-related hormones in the chum salmon pituitary (Kawauchi et al., 1980a, b, c, 1981, 1982), a large amount of basic protein has been isolated from an acid acetone extract of the pituitaries. The protein was identified as a PRL by its biological and immunohistochemical characteristics. This paper describes the purification procedure, and physicochemical and biological properties of the salmon PRL, together with partial amino acid sequence.
CHUM SALMON
MATERIALS
AND METHODS
Pz!rific&ion. Whole pituitary glands were taken from mature female chum salmon, Oncorhynchrrs lieta. caught in Tsugaruishi river. Iwate, Japan during the month of December. They were frozen immediately in liquid nitrogen. Routinely 50 g of the pituitaries were extracted with 150 ml of acid acetone (cont. HCl:acetone, 1:28 v/v) at 0” for 1 hr and the residue was reextracted with 100 ml of 80% acetone at 0” for 1 hr. The extract was precipitated by addition to acetone (3 liters) prechilled for 24 hr at 4”. The precipitate was submitted to gel filtration on Sephadex G-25 (medium) column (5.5 x 68 cm) in 0.1 N acetic acid. The unretarded fraction was lyophilized and dissolved in distilled water (100 ml) at pH 3.0. The pH of the solution was adjusted to 4.4 with 0.1 N NaOH. The resulting precipitate was removed by centrifugation and the supernatant was introduced into a column of CM-Sephadex C-25 (1 x 30 cm), equilibrated with 0.05 M ammonium acetate, pH 4.6. After washing with the initial buffer, elution was performed with a gradient formed by passing 0.2 M ammonium acetate, pH 9.0, through a mixing flask containing 200 ml of the starting buffer. The PRL fraction was finally purified on a Sephadex G-100 column (2.64 x 47 cm). equilibrated with 0.05 M ammonium acetate, pH 6.8. Chetnicnl
and
physicochemical
characterization.
The purified PRL was examined by slab gel electrophoresis at pH 4.3 and 7.5 in 7.5% polyacrylamide gel (Grnstein, 1964) stained with 0.25% Coomassie brilliant blue R 250 in methanol-acetic acid-water (5: 1:5, v/v) (Meyer and Lamberts, 1965) and by gel isoelectric focusing in 5% polyacrylamide gel at 2% ampholine (pH 3.5-10 and pH 7-11) (Wrigley, 1971), stained with 0.04% Coomassie brilliant blue G 250 in 3.5% HCIO, (Reisner et a/.. 1975). The isoelectric point was estimated by gel isoelectric focusing with amphoiine (pH 7- 11). Protein samples containing PI-marker proteins (Oriental Yeast Co.) were focused in duplicate disc gels. One of the gels was stained and the other was sliced in 0.5 cm segments. The pH of each slice was measured after agitation in i ml of distilled water for 2 hr. The molecular weight was estimated by sodium dodecyl sulfate (SDS) slab gel electrophoresis (Weber and Osborn. 1969) and by exclusion chromatography on Sephadex G-100 using 0.1 1M Tris-HCl, pH 8.2. Egg white albumin, chymotrypsinogen, and lysozyme (Boehringer-Mannheim), and molecular weight markers (14,300-71,500) (BDH Chemicals) were employed as reference standards. Circular dichroism spectra were taken in a Gary model 60 spectropolarimeter, equipped with a Model 6002 circular dicbroism attachment in 0.1 M Tris-HCI, pH 8.2. The content of a-helix was estimated as described by Bewley et al. (1969).
PROLACTIN
447
An absorptivity value of the salmon PRL. pmc ,cm,esOnmwasdetermined to be 0.306 based on weight assuming a 15% moisture content. Protein concentrations were determined from absorption spectra after correction for light scattering as described by Beaven and Holiday (1952). For amino acid analysis, the protein was hydrolyzed for 22 hr at 110” with constant boiling HCl. The analysis was carried out with an I.323 automated amino acid analyzer (Type 4400). NH,-Termina? analysis was performed by the dansyl method (Gray, 1967) and COOH-terminal residue was determined by amino acid analysis of hydrazinolysate of performic acid oxidized PRL: according to Akabori et al. (1956). The amino acid sequence of the intact hormone was determined by the fluorescein-isothiocyanate as described previously (Muramoto et al., 1978). The fluorescein-thiohydantoin amino acids were identified by thin-layer chromatography on polyamide sheets. For further determination of the amino acid sequence, the protein was treated with cyanogen bromide, followed by performic acid oxidation. The resulting peptides were fractionated by gel filtration GII Sephadex G-50 in 0.1 N acetic acid and Sephadex G-75 in 0.1 N acetic acid, by droplet countercurrent chromatography on DCC-A (Tokyorika Co.) with !Ibutanol:pyridine:0.6 Iw ammonium acetate (pH 6 8) (5:3: 10, v/v), and high voltage paper electrophoresis in formic acid-acetic acid, pH 1.9 at 2000 V. Each fragment was digested by trypsin, chymotrypsin. and thermolysin. and subjected to high-performance liquid chromatography on reverse phase column C,, with 10 n&’ ammonium acetate (pH 4.0) and isopropanal. The amino acid sequence of these peptides were determined by the dansyl Edman procedures (Bruton and Hartley, 1970). Biologicai cizaracterizarion. Sodium-retaining activity of the salmon PRL was examined using intact tilapia and rainbow trout. The assay protocol for tilapia. Sarotkerodon mossambicus was the same as described by Clarke (1973). except that smaller fish weighing 1.5-3 g were used in the present study. They were acclimated to seawater (24 i- 1”) for more than 3 weeks before use. Ovine PRL (NIAMDD o-PRL-i3) was used as a reference standard. The fish were pretreated with 2 ,&g of ovine PRL for 3 days and subsequently given intraperitonial injections of saline (0.9% NaCI) or PRLs for 2 days. They were sacrificed 24 hr after the last injection. Blood samples were taken from the caudal artery following the method described by Jozuka and Adachi (1979). Similarly. assays v.ere carried using juvenile rainbow trout, S&no gnivd~eri, weighing 5-10 g. They were placed in 33% seawater for 3 days and then transfered to 50% seawater. Those fish acclimated to 50% seawaler for more than 7 days were injected intraperitoneally with stirnon or ovine PRL, and sacrificed 24 hr after the injection. Aiiquots
448
KAWAUCHI
of plasma (1 ~1 each) were diluted to 1 ml with deionized water and Na concentration was determined by atomic absorption spectrophotometer (Hitachi 208 or 180). Data are presented as means t SE. Multiple nonindependent comparison (Dunnett’s procedure) was applied to compare the means with a control. Immunochemical characterization. Antisera were raised against the salmon PRL in rabbits. The antigen (1 mg) was dissolved in 0.9% NaCl (1 ml) and emulsified with complete Freund’s adjuvant (1 ml). Seven injections, 1 mg each, were given subcutaneously into the foot pads and the back at intervals of 3 weeks. They were bled 2 weeks after the last injection, and the sera were lyophilized. The sera were tested by Guchterlony technique (Ouchterlony, 1953). Cross reactions of the antisera with ovine and whale PRLs (Kawauchi and Tsubokawa, 1979) and pituitary extract of tilapia were tested by agar diffusion techniques. An immunoglobulin (IgG) fraction was prepared from the antisera by ammonium sulfate fractionation. The antisalmon PRL rabbit IgG was used for immunocytochemical identification of PRL secreting cells in the chum salmon pituitary by peroxidase-antiperoxidase method (Sternberger et al., 1970). Details of this procedure will be described elsewhere (Naito et al., in press).
RESULTS
Chemical and physicochemical properties. The acid acetone extract of the salmon pituitary was subjected to gel tiltration on Sephadex G-25 to separate smaller peptides, such as endorphins and melanotropins from larger proteins as described previously (Kawauchi et al., 1980~). The unretarded fraction contained a large amount of NH,terminal peptide of salmon proopiocortins, which were removed by isoelectric precipitation (Kawauchi et al., 1981). As shown in Fig. 1, the supernatant consisted mainly of prolactin. When subjected to gel filtration on Sephadex G-100, the salmon PRL emerged as a single symmetrical peak with VJV, = 2.0. A yield of 50 mg was obtained from 50 g (wet weight) of the pituitaries. Polyacrylamide gel electrophoresis at pH 4.3 revealed only one intensely stained band, whereas no band was detected at pH 7.5. The isoelectric focusing of the salmon PRL exhibited a single band at pH 3.5% 10, and one intensely stained major band with a
ET AL.
second faint band running immediately behind it at pH 7- 11 (Fig. 2). The isoelectric point of the main band was estimated to be 10.3 (Fig. 3). The molecular weight of the salmon PRL was determined to be 22,300 by SDS polyacrylamide gel electrophoresis and 23,400 by exclusion chromatography on Sephadex G-100 (Fig. 4). The amino acid composition of the salmon PRL was calculated on the basis of a molecular weight of 22,300. As shown in Table 1, the total number of the residues by this calculation was 194, which is comparable to that in ovine PRL but exceeds tilapia PRL by 20 residues. A relatively high content of basic amino acids and low content of Glu are consistent with the high value of the isoelectric point. It is to be noted that the salmon PRL as well as tilapia PRL consisted of 4 half-cystine residues, which is in contrast with 6 residues in mammalian PRLs. As with tilapia PRL, relatively low content of tryptophan and tyrosine made the extinction coefficient low, 0.306. The high content of serine and methionine may be characteristic of salmon PRL. Isoleucine was identified as a sole amino-terminal residue by the dansyl method. The NH,-terminal sequence as determined by analysis of the intact hormone was H-Ile - Gly - Leu - Ser - Asp Leu - Met - Glu - Arg - Ala - . Hydrazinolysis of the intact hormone gave no amino acid, whereas that of the performic acid oxidized hormone yielded cysteic acid. Thus the COOH-terminal residue turned out to be half-cystine as are mammalian and tilapia PRLs. The salmon PRL gave 10 peptide fragments by cyanogen bromide cleavage and performic acid oxidation after fractionation using gel filtration, droplet countercurrent chromatography, and high voltage paper electrophoresis. The amino acid composition of these peptides is summarized in Table 2 and the total residues are consistent with that of the intact hormone. By
CHUM
SALMON
449
PROLACTIN
-------_-------
O*fq,~,O
_jt_
~X'~
QJN---..-
ACETATE
FIG. 1. Ion-exchange chromatography of the salmon PI& fraction on CM-Sephadex C-25. Initially, acid acetone extract of the salmon pituitary (50 g) was chromatographed on Sephadex G-25. The first fraction (390 mg) was subjected to isoelectric precipitation to remove a predominant acidic peptides: such as amino-terminal peptide of proopiocotrins (Kawaucbi et al., 1981). The supernatant was introduced to a column of CM-Sephadex C-25 (1 x 30 cm). Elution was performed with a gradient formed by passing 0.2 M ammonium acetate, pH 9.0, through a mixing flask containing 200 ml of the starting buffer at 3 ml/l0 minifraction.
analyzing amino acid sequence of the fragments as well as the intact hormone, CB-6 was identified to be the NH,-terminal peptide, followed by CB-5. It is evident that the salmon PRL lacks the NH,-terminal disule loop of the mammalian PRLs. CB-3 was assigned to the COOH-terminal peptide, due to the presence of half-cystine residue and absence of methionine at the carboxyl-terminal. CR-2 exhibited significant sequence homology with the COOHterminal portion of mammalian PRLs and GHs, especially of human PRL (172- 192). The partial amino acid sequence is aligned with the structures of mammalian PRLs and
GHs in Fig. 5. etails of studies on the mary structure will be described elsew The circular dichroism spectra are s in Fig. 6. The salmon negative band at 221 and which are characteristic of an a-helix. a-helix content was about 50% to nearest 5%. The spectrum in t side chain absorption exhibite peak at 291 nm with a shoulder around nm, a crossover point at 276 nm weak, negative shoulders near 267 nm. Biological
retaining
properties.
activity
The
of the salmon
s
pri-
the
300 two
261
450
KAWAUCHI
FIG. 2. Gel electrophoretic patterns of the purified salmon PRL. (A) Slab gel electrophoresis at pH 4.3; 50 pg on 7.5% gel stained with Coomassie brilliant blue R 250. (B) Gel isoelectric focusing at 2% ampholine (pH 3.5- 10) and (pH 7- 11); 50 pg each on 5% polyacrylamide stained with Coomassie brilliant blue G 250.
shown in Table 3. When tested in tilapia, the salmon PRL was inactive even at a dose of 20 pg/g, although the plasma sodium increased significantly after ovine PRL injection at 20 pgig. When injected into rainbow
ET AL.
0
0.4
0.6 MOBILITY
0.8
a
Lysozyme(l4600)
0.6 -
Salmon
r ; z % 0 .4-
PRL(22300) 0 = 2.0 f
(42900) 2 .0-
Salmon PRL(Z3400) Chymotrypsinogen (25000)
\
O-
1.5 4.0
4.5
5.0
Log nw
A
B
4. Determination of the molecular weight of the salmon PRL. (A) SDS gel electrophorcsis using molecular weight markers (14,300-71,500) obtained from BDH Chemicals (England). (B) Exclusion chromatography on Sephadex G-100 in 0.1 M Tris-HCl, pH 8.2. FIG.
, 1.0
FIG. 3. Estimation of the isoelectric point of the salmon PRL. The salmon PRL was focused in duplicate disc gel with ampholine (pH 7- 11). One of the gels was sliced in 0.5~cm segments for pH measurement, and the other was stained with Coomassie brilliant blue G 250. The pZ marker proteins consisted of cytochrome c from horse and the acetylated cytochromec; pZ, 6.4,8.3,9.7,and 10.6(OrientalYeastCo.).
2.5
0.s-
0.2
CHUM SALMON TABLE 1 AMINO Aclr, COMPOSITION OF SALMON PRL COMPARED WITH TILAPIA AND OVINE PRLs Amino acid
Salmonm PRL
Tilapia* PRL
Asp Thr Ser ml Pro Cl)’ Ala Cysi? Val Met Ble Leu TYr Phe Trp His LYS Arg
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 6.6 i3 .O 12.4
16.4 9.4 21.6 17.4 10.8 8.1 9.6 4.2 6.8 5.4 9.3 24.5 3.0 4.7 1.0 4.9 8.9 6.8
Ovine< PRL 22 9 15 22 11 11 9 6 10 7 11 23 7 6 2 8 9 11
PROLACTIN
45i
chum salmon pituitary with anti-salmon PRL rabbit hgGI showed strong cross reaction only in follicle forming cells of the rostral pars distalis at a dilution of 114000 (Figs, 8 and 9). These cells correspond with the PRL cells of chum salmon identified with light and electron microscopy (Nagahama, 1973): and distinguished from the GEL cells in the proximal pars distalis. No specific reaction was observed between the antibody and the GIL cells at any concentration tested. BISCUSSI0N
During the course of our studies on characterization of proopio~ort~~-~e~~te~ hormones from chum salmon pituitaries, prolactin was isolated as one of the predominant components from aci extract of the pituitary. Extraction by acid Total 193.4 172.8 199 acetone has been employed for isolation of mammalian PRLs as well as ACTHs and 0 Values were calculated on the basis of the molecular weight of 22,300. Threonine and serine values P-LPHs (Cole and Li, 1955) and proved to were corrected for destraction. Tryptophan was be an excellent procedure for obtaining determined by spectrophotometric method (Donovan, highly purified whale PRL and GE (Ka1969) and by methane sulfonic acid hydrolysis. wauchi and Tsubokawa, 1979; Tsubokawa b Taken from Farmer et al. (1977). et al., 1980). In contrast, fish irri3ve c Taken from Li et al. (1970). ctior; been prepared exclusively by at neutral or alkaline conditions. In such conditions, however, several undesirable trout acclimated to 50% seawater, both salmon and ovine PRLs caused significant side reactions may take place: enzymatic increase in plasma sodium after 24 hr, and 1 degradation will occur at neutral ~14, as ILg/g of the salmon PRL gave a similar re- well as deamidation and aggregation at sponse with 12.5 @gig of ovine PRL. alkaline pM. In addition, the chum salmon lmmurzoclzemical properties. Figure 7 PRL was proved to be a strongly basic shows an Ouchterlony agar diffusion plate protein and may not be extractable effiwith anti-salmon PRL rabbit serum in the ciently in alkahne condition. center well and ovine and whale PRLs and Most of the mammalian P the pituitary extract of tilapia in the tions have revealed several bands on pslyperipheral wells. A single strong precipitin acrylamide gel electrophoresis, and some of line was obtained between the antiserum the bands are assumed to be deamidated forms. The salmon PIPE exhibited only one and the salmon PRL. The pituitary extract of tilapia gave a reaction of partial identity electrophoretic band at pH 4.3 and in am3.5- IO) but a very faint and with the salmon PRL, but no line was observed between the antiserum and mammaslightiy acidic band in ampholine (pK lian PRLs. 7- 11). The minor component could be a Immunocytochemical staining of the deamidated form as are the cases Em mam-
452
KAWAUCHI
ET AL.
TABLE 2 AMINO ACID COMPOSITION OF CYANOGEN BROMIDE (CB) FRAGMENTS OBTAINED FROM SALMON PRL Amino acid Asp Thr Ser Glu Pro GUY Ala Cysl2” Val Met* Ile Leu Tv Phe His LYS Arg Trp Totald NHz-terminal residue
CB-1
CB-2
CB-3
9.6 2.2 10.6 8.3 6.2 3.3 4.0 1.5 2.0 + 4.8 13.9 1.0 0.4 1.9 6.1 2.9 +’
1.9 1.0 3.1
1.0
1.7 0.9 2.8 3.7
1.0
+ 2.9 3.8 0.8 3.0 1.0 2.0 0.9
(81)
24
5
33
28
7
Ser
A%
GUY
Glu
Ile
ND
1.0 0.9 0.7 2.0 0.8 + 0.6 1.6
CB-4
CB-5
CB-6
4.0 0.9 4.5 1.3 2.2 3.7
3.4 1.5 4.8 2.4 2.2
1.1
CB-7
CB-8
CB-9
CB-10
0.9
0.7
0.9 2.4
1.1
1.0
1.1 1.0 -
+ 4.0
+ 1.0 2.1
1.0 1.6 2.4 2.3
1.1 +
1.4 +
+
+
1.0 0.8 4 GUY
0.9 4
5
1
Val
Pro
Met
Note. The salmon PRL was treated with cyanogen bromide, followed by performic acid oxidation. The resulting peptides were fractionated on Sephadex G-50 and G-75 in 0.1 N acetic acid. The smaller peptides were separated by high voltage paper electrophoresis in formic acid-acetic acid, pH 1.9. The larger peptides were purified by droplet countercurrent chromatography with a solvent, n-butanol:pyridine:0.6 M ammonium acetate (pH 6.8) (5:3:10, v/v). a Determined after performic acid oxidation. b Homoserine + hormoserine lactone. r Detected by the uv absorption. d The values were obtained by sequence analysis except for CB-1.
malian PRLs. The isoelectric point of the salmon PRL (10.3) was notably higher than those of mammalian PRLs; p1 of ovine PRL is 5.6. An isoelectric point of the chum salmon PRL prepared by Idler et al. (1978) was reported to be 6.0. In contradiction with such a low isoelectric point, however, the protein was not absorbed on the anion exchanger of DEAE Bio Gel at pH 9.0. According to Farmer et al. (1975), tilapia PRL gave no electrophoretic band at pH 8.3. The same authors claimed later than a faintly stained band with an Rf of
band at pH 8.3 could not be a major but a minor or deamidated component. It seems more likely that the tilapia PRL as well as the salmon PRL is too basic to enter a gel at a slightly alkaline pH rather than that the protein is unstainable on the gel. The molecular weight of the salmon PRL was estimated to be 22,300, which is 3000 larger than that of tilapia PRL and similar to that of ovine PRL. Total amino acid residues were estimated to be 194, and amino acid analysis of ten cyanogen bromide fragments supports the estimation. The salmon PRL possessed four halfcystine residues or two disulfide bridges, as in the case of tilapia PRL. It has been speculated that the amino-terminal disulfide
Asp-Thr-Phe
0-Gti:
Glu-Phe-G1u-Arg-;h5r-Tyr-Ile
z!u-Phe-Glu-Glu-Ala-?&Ile
&$..His-Phe-Pro
Pro i-7 G,"-G,y-Gln-Arg--------------------------Leu-Le~-
( )-(
)-Arg-Phe-~8~-Glu-Ala-Ser
)-3:5--GlbGly-(
A?~-we-cooti
l;?;-Phe-COON
)-Ser
COOH
( )-Ser-(
)-Ile-His-a~~-Asn-Asn
coot4
coon
)-(
Ile-(
)--Ile-~:~-Aso-Am-Asn
Ala-Thr-Lys-Met-F&Pro-Gin-Thr
)-(
I?@-(
M',:-------------------------------------------------------------------------------------
NH2-Phe-Pro-Thr
NH,-Ala-Phe-Pro-Ala
FIG. 5. Partial amino acid sequence of the salmon PRL in comparison with mammalian PRLs and growlh hormones (GHs). The amino acid sequence of the salmon PKI. had been determined up to 35 residues at the NH,terminal and 30 residues at COOH-terminal. To &kin maximum homology with the other PRLs and CIIIs, the NIIYterminal residue of the salmon PRL was aligned with the 13th residue of the manmnlian PI&s. Three half-cystine residues at C~OF~-t~rrn~ll~i region were aligned in homologous positions to those of the mammalian hormones. The amino acid scqtlences are taken from ovine PRL (Li et al., 1970), human PKL (Shomc and Parlow, 15X77), ovine 611 (Li CT rrl., 1973); and human GH (Li, IY72).
Ser-Leu-5
Nt~,-Ceu-Pro-lle-~Cyz-Pro-Gly-Gly-Ala~-Ala-Arg-Cys-Gln
N~,-Thr-Pro-.Val-Cys-P:o-Asn-Gly-Pro-Gly-~~~-Cy~-Gl~-V~l~S~r NW,-Thr-Pro-.Val-Cys-P:o-Asn-Gly-Pro-Gly-~~~-Cy~-Gl~-V~l~S~r
S-PRL:
GH:
GH:
PRL:
Salmon
Ovine
PQL:
Hunan
HUIUII
PRL:
Ovine
z
F c: z! 2
z"
454
KAWAUCHI
ET
AL.
WAVELENGTH ( nm )
A
B
FIG. 6. Circular dichroism spectra of the salmon band absorption: (B) the side chain absorption.
PRL
bond in mammalian PRLs is absent in tilapia PRL, mainly because the aminoterminal tetrapeptide of the tilapia PRL revealed no half-cystine residue. In addition, selective reduction of the amino-terminal disulfide bond of ovine PRL increased the biological activity of the hormone, when assayed on permeability change in Gillichthys urinary bladder (Doneen et al.,
Fish
ACTIVITY
OF OVINE
Hormone
Tilapia
Saline o-PRL
s-PRL
Rainbow trout
Saline o-PRL
AND
SALMON
3 PRLs
Dose ~/-&g/g bw)
different
from
-
8 8 8 8 8 7 8
1
0.2 1 2.5 saline-injected
Number fish
5 10 20 0.5 5 20
5 12.5
* Significantly
IN TILAPIA
-
s-PRL
control
pH 8.2. (A) The amide
1979). As shown in the present studies, the maximum homology was found between the salmon PRL and mammalian PRLs and GHs, when the amino-terminal residue of salmon was aligned with the 13th residue of mammalian PRL. Three of the four halfcystine residues were identified at the carboxy1 terminal portion and aligned in the homologous positions with those of mam-
TABLE SODIUM-RETAINING
in 0.1 M Tris-HCl,
at P < 0.05.
of
AND
RAINBOW
TROUT
Plasma Na (mmoYliter) (mean i SE) 167 172 173 193 167 160 168
f IL * + t ? f
2.2 3.4 3.6* 6.0* 2.2 4.0 2.3
162.1 172.8 176.3 183.1 168.6 182.4 194.4
2 2 f + k -c -t-
4.7 5.4 2.7* 3.7* 3.2 2.9* 5.6*
CHUM SALMON
PROLACTlN
455
evidence for a common ancestral molecule for PRLs and GHs. Besides the similarity in the overall molecular features with the mammaIia~ hormones, the protein obtained in the present study was identified as by its immunological and biologica vities. Various hormone-secreting cells of the chum salmon pituitary have been identified by light and electron microscopy (Nagabama, 1973). The PRL cells in the chum salmon are arranged in the form of follic form the bulk of the rostra1 pars whereas GH cells are found ex~l~~ivel~ in the proximal pars distalis. In the present study, incubation of chum salmon pituFIG. 7. Immunodiffusion plate showing precipitin itaries with anti-salmon P reactions of salmon PRL, ovine PRL, whale PRL, and strong reaction only in the follicular pituitary extract of tilapia with the anti-salmon PRL cells, and no other cells in the pituitary rabbit serum in the center well. Photographed after 2 this reaction. In addition, pituitary extract hr of diffusion. of tilapia reacted with anti-salmon PRL in agar diffusion test, although precipitin lines malian hormones. Thus the salmon PRL hete definitely lacks one disulfide loop at the suggest immunological amino-terminal portion. However, it also partial identity) of these fish seems possible that the structure of the cular dichrois data, however, conformational similarity between the salprohormone of the salmon PRL is similar with that of mammalian PRL, and that the mon and tilapia P Ls: the side chain absorption spectra w e remarkably identical. processing of the prohormone could make of P entification the *‘phenotype” different. The resemblance of primary structure between mammalian PRLs and GHs the tilapia PRL antibodies bound strongly suggested that they have evolved RL and GH cells in two species of from a common ancestral molecule (Bewley s (Nagahama et al., 198I). and Li, 1971). In addition tilapia PRL as It has been well established im many telewell as some reptilian and amphibian PRLs ost species that PRL helps restore sodi have been shown to have some biological and immunochemical GH activities (Far- balance in fresh water by rnai~~a~~i~~ mer et nl., 1975; Nicoll and Licht, 1971; passive loss component throu a low level. On the ot Hayashida et nl., 1973). In our unpublished ment of fish in salt wa observation, the salmon PRL was imity to excrete sodium ions, t m~~olQgically active and parallel with of plasma sodium levels tilapia GII in two radioimmunoassay sys- an elevation (Clarke and Bern, 1980). Idler et al. (19778) tem employing rat GH antiserum (Hayashida, 1970) and snapping turtle GH an- have isolated salmon P retaining action in hyp tiserum (I-Iayashida et al., 197.5), although the activity was low. The sequence com- Poecilin. In the present s L was lo- IS times as active as ravine parison (Fig. 5) seems to provide further
456
KAWAUCHI
ET
AL.
FIG. 8. Sagittal section of Oncorhynchzrs beta pituitary stained with an anti-salmon PRL rabbit IgG at a concentration of 114000, counterstained with Meyer’s hematoxyline. RPD. rostra1 pars distalis; PPD, proximal pars distalis; PI, pars intermedia. x22. FIG. 9. Sagittal section of the rostra1 pars distalis of 0. keta pituitary stained with an anti-salmon PRL rabbit IgG at a concentration of 114000 counterstained with Meyer’s hematoxyline. Immunoreactivity is evident in PRL cells, whereas no reactivity is seen ACTH cells and GH cells. x264.
PRL in increasing plasma sodium of 50% seawater-acclimated rainbow trout, suggesting that its molecular structure is considerably specialized for an osmoregulatory role in salmonids. Similarly, tilapia PRL has been found to be approximately 100 times as active as ovine PRL in the tilapia sodium-retaining bioassay (Farmer et al., 1977). However, the salmon PRL was not active in tilapia assay, although ovine PRL was effective, indicating species specificity of fish PRLs. Absence of the tilapia sodium-retaining activity of the salmon PRL is in good accord with the original findings that pituitary extracts of salmonid
fishes were without effect in tilapia (Clarke, 1973). We have also tried to develop a homologous sodium-retaining assay using chum salmon fry adapted to seawater. However, salmon as well as ovine PRLs were without effect even though larger doses of the hormones were injected repeatedly. At any rate, sodium-retaining assay using rainbow trout as in the present study is far from satisfactory as a bioassay for fish PRL. Further studies are needed to increase accuracy and validate specificity of the assay in order to provide conclusive evidence for the physiological role of the salmon PRL.
CHUM
SALMON
ACKNOWLEDGMENTS We thank Hiroto Okamura and Ryutaro Kudo for their skilled technical assistance. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture to H. K. and T. H.. and also from the Japan Society for Promotion of Science to T. H.
REFERENCES Akabori, S., Ohno, K.. Ikenaka, T., Okada, Y., Hanafuss, H., Haruna, I.. Tsugita, A., Sugae, K., and Matsushima, T. (1956). Hydrazinolysis of peptides and proteins. II. Fundamental studies on the determination of the carboxyl ends of proteins. Bull. Chern. Sm. J~~purr 29, 507-518. Beaven, 6. H., and Holiday, E. R. (1952). Ultraviolet absorption spectra of proteins and amino acids. Atl~rn. Protein Chem. 7, 319-386. Bern. II. A. (1975). Prolactin and osmoregulation. Amer. Zool. 15, 937-948. Bewley. T. A., Brovetto-cruz, .I., and Li, C. H. (1969). Human pituitary growth hormone. Physicochemical investigations of the native and reducedalkylated protein. Biochemisrry 8, 4701-4708. Bewley, T. A., and Li, C. H. (1971). Sequence comparison of human pituitary growth hormone, human chorionic somatomammotropin and ovine pituitary lactogenic hormone. Experierriia 27, !368- 1371. Bruton, C. J.. and Hartley. B. S. (1970). Chemical studies on meihionyl-tRNA synthetase from Escherichicr coli. i. Mol. Bio/. 52, 1655 178. Clarke, W. C. (1973). Sodium-retaining bioassay of prolactin in the intact teleost Ti/apia mossambica acclimated to seawater. Gen. Conzp. .%ducvinol. 21, 498-512. Clarke. W. C.. and Bern, H. A. (1980). Comparative endocrinology of prolactin Irr “Hormonal Proteins and Peptides” (C. H. Li. ed.), Vol. 8, pp. iCt- 197. Academic Press, New York. Cole. R. D.. and Li. C. H. (1955). Studies on pituitary lactogenic hormone. XIV. A simplified procedure of isolation. J. Biol. Che/?l. 213, 197-201. Doneen: B. A., Bewley, T. A., and Li, C. H. (1979). Studies on prolactin. Selective reduction of the disulfide bonds of the ovine hormone. Biochemistry 18, 4851-4860. Donovan. .I. W. (1969) Ultraviolet absorption. 112 “Physical Principles and Techniques of Protein Chemistry” (Leach. S. J., ed.), Part A, pp. IOl- 170. Academic Press, New York. 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. Lv‘e SC?. 16, 1499 i.58. Farmer: S. W.. Papkoff: H.. Bewley. T. A..
PROLACTIN
457
Hayashida, T., Nishioka, R. S., Bern, II. A., and Li, C. H. (1977). Isolation and proper?ies of teieost proiactin. Getz. Camp. Endocrinoi. 31, M-71. Gray. W. R. (1967). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. H. W. Hirs, ed.); Vol. II, pp. 469-475. Academic Press, New York. Hayashida. T. (1970). Immunoiogicai studies with rat pituitary growth laormone (RGH) II. Comparative immunochemical investigation of GH from repre sentatives of various vertebrate classes with monkey antiserum to RGH. Gen. Camp. Enciooiricii 15,432-452. Hayashida, T.. Licht. P., and Nicoll, C. S. (1973). Amphibian pituitary growth hormone and proiactin: Immunochemical relatedness to rat growth hormone. Science 182, 169- 171. Hayashida, T.. Farmer, S. W.. and Papkoff, H. ( 1975). Pituitary growth hormones. Further evidence for evolutionary conservatism based on immunochemical studies. Proc. Wnr. i\cati. Sci USA 72, 4322-4326. Idler, I), R., Shamsazzaman, K. M., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Gerr. Camp. Etzdocrinoi. 35, 409-438. Jozuka, K., and Adachi, II. (1979). Environmenta! physiology on the pH tolerance of teleost. II. Blood properties of medaka. Orr:irr.\ laf;pc.x. ex-posed to low pI5 environment. At~lrof. Zool. Jupun 52, 107-113. Kawauchi. H., and Tsubokawa, M. (1979). Isolation and characterization of fin whale prolactin. ir!t. .i. Pepride Proreirr Res. 13, 229-234. Kawauchi, II., Tsubokawa, M.. Kanerawa. A., and Kiiagawa, H. (1980a). Occurrence oftwo different endorphins in the salmon pituitary. Biocherrr. Biophys. Rrs. Cornmnn. 92, 127% 1288. Kawauchi. II.: Ada&i, Y., and Tsubokawa. M. (1980b). Occurrence of a new melanocyte stimulating hormone in the salmon pituitary glands. Biochem. Bi0ph.v~. Res. Ctinim~i2. 90, 1508- 1517. Kawauchi, H.. Adachi. Y.. and Ishizuka, B. (I%&). Isolation and structure of another /3-melanotropin from salmon pituitary glands. Int. J. Prplitic Protein Res. 16, 79982. Kawauchi, II.. Takahashi, A., and Abe. K. (1981). Gamma-melanotropin is not present in an 2% terminal peptide of salmon proopiocortin. 1111. /. Peptide Protein Res. 18, 223-727. Kawauchi, II., Takahashi, A., and Abe. Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pimitary glands. ArcA. Biochem. Biophyr. 213, 680-688. Li, C. H., Dixon, 3. S.. Lo. T-B.. Schmidt, K. D., and Pankov. Y. A. (1970). Studies on pituitary iactogenic hormone. XXX. The primary strucrcre cf
458
KAWAUCHI
the sheep hormone. Arch. Biochem. Biophys. 141, 705-737. Li, C. H. (1972). Hormones of the adenohypophysis. Proc. Amer. Phil. Sot. 116, 365-382. Li, C. H., Gordon, D., and Knorr, J. (1973). Primary structure of sheep pituitary growth hormone. Arch. Biochem. Biophys. 1.56, 493-508. Meyer, T. S., and Lamberts, B. L. (1965). Use of Coomassie brilliant blue R 250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide gel strips. Biochim. Biophys. Acta, 107, 144- 145. Muramoto, K., Kawauchi, H., and Tsuzimura, K. (1978). Sequence determination of peptide by the combined use of fluorescein isothiocyanate and phenylisothiocyanate. Agric. Biol. Chem. 42, 1559- 1563. Nagahama, Y. (1973). Histophysiologica! studies on the pituitary gland of some teleost fishes, with special reference to the classification of hormone-producing cells in adenohypophysis. Mem. Far. Fish. Hokkaido Univ. 21, l-63. Nagahama, Y., Olivereau, M., Farmer, S. W., Nishioka, R. S., and Bern, H. A. (1981). Immunocytochemical identification of the prolactinand growth hormone-secreting cells in the teleost pituitary with antisera to tilapia prolactin and growth hormone. Gen. Comp. Endocrinol. 44, 389-395. Naito, N., Takahashi, A.. Nakai, Y., Kawauchi, H., and Hirano, T. “Immunocytochemical Identification of the Prolactin-Secreting Cells in the Teleost Pituitary with Antiserum to Chum Salmon Prolactin.” Gen. Comp. Endocrinol., in press. Nicoll, C. S., and Licht, P. (1971). Evolutionary biology of prolactins and somatotropins. II. Electrophoretic comparison of tetrapod somatotropins. Gen. Comp. Endocrinol. 17, 490-507.
ET AL. Ng, T. B., Idler, D. R., and Bruton, M. (1980). A comparison of prolacting from a marine, an estuarine, and a freshwater teleost. Gen. Comp. Endocrinol. 42, 141- 146. Ornstein, L. (1964). Disk 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. Reisner, A. H., Nemes, P., and Bucholtz, B. (1975). Use of Coomassie brilliant blue G 250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64, 509-516. Shome, B., and Parlow, A. F. (1977). Human pituitary prolactin (PRL): the entire linear amino acid sequence. .I. Clin. Endocrirlol. Metab. 45, 1112-1115. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., and Meyer, H. G. (1970). The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. .I. Histochem. Cytochem. 18, 315-333. Tsubokawa, M., Kawauchi, H., and Li, C. H. (1980). Isolation and partial characterization of growth hormone from fin whale pituitary glands. J. Biochem. 88, 1407- 1412. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. Wrigley, C. W. (1971). Gel electrofocusing. In “Methods in Enzymology” (W. B. Jakoby, ed.), Vol. 22, pp. 559-564. Academic Press, New York.
AND
COMPARATIVE
Isolation HIROSHI
School
49,
4466458 (1983)
and Properties
of Chum
ENDOCRINOLOGY
KAWAUCHI, KEN-ICHI ABE, AKIYOSHI SANAE HASEGAWA,” NOBUKO NAITO,?
of Fisheries University
Sciences, of Tokyo,
Kitasato Nakano,
Salmon
Prolactin
TAKAHASHI, TETSUYA HIFUNO,* AND YASUMITSU NAKAI?
University, Sanriku, Iwate 022-01; Tokyo 164, and tSchoo1 of Medicine Shinagawa, Tokyo 142. Japan
*Ocean Shown
Research University,
Institute,
Accepted April 19, 1982 A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mgig of wet tissue. It was lo- 1.5times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with VJV, = 2.0. Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an o-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH,-terminal residue, isoleucine, and a single COOH-terminal residue, halfcystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH,-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.
It has been suggested that prolactin (PRL) and growth hormone (GH) have evolved from a common ancestral molecule, based mainly on structural similarities between the mammalian hormones (Bewley and Li, 1971). PRLs are known to exhibit multiple actions among vertebrates, whereas GH is exclusively concerned with stimulation of animal growth (Bern, 197.5; Clarke and Bern, 1980). To obtain better insight into the evolutionary history of these hormones and the great diversity of their functions, it is essential to isolate and characterize PRLs and GHs at many phylogenetic levels. When compared with our knowledge of the chemistry of mammalian PRLs, little is known about the nonmammalian hormone. Earlier studies to isolate fish PRL had limited success because of the absence of a convenient bioassay (Clarke and Bern, 446 00 16-6480/83/030446- 13$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
1980). Tilapia PRL has now been isolated using a sodium-retaining assay and extensively characterized physicochemically (Farmer et nl., 1977). Recently, isolation and partial characterization of fish PRLs have been reported using salmon (Idler et al., 1978), carp and flounder (Ng et al., 1980). During the course of our studies on purification and characterization of the proopiocortin-related hormones in the chum salmon pituitary (Kawauchi et al., 1980a, b, c, 1981, 1982), a large amount of basic protein has been isolated from an acid acetone extract of the pituitaries. The protein was identified as a PRL by its biological and immunohistochemical characteristics. This paper describes the purification procedure, and physicochemical and biological properties of the salmon PRL, together with partial amino acid sequence.
CHUM SALMON
MATERIALS
AND METHODS
Pz!rific&ion. Whole pituitary glands were taken from mature female chum salmon, Oncorhynchrrs lieta. caught in Tsugaruishi river. Iwate, Japan during the month of December. They were frozen immediately in liquid nitrogen. Routinely 50 g of the pituitaries were extracted with 150 ml of acid acetone (cont. HCl:acetone, 1:28 v/v) at 0” for 1 hr and the residue was reextracted with 100 ml of 80% acetone at 0” for 1 hr. The extract was precipitated by addition to acetone (3 liters) prechilled for 24 hr at 4”. The precipitate was submitted to gel filtration on Sephadex G-25 (medium) column (5.5 x 68 cm) in 0.1 N acetic acid. The unretarded fraction was lyophilized and dissolved in distilled water (100 ml) at pH 3.0. The pH of the solution was adjusted to 4.4 with 0.1 N NaOH. The resulting precipitate was removed by centrifugation and the supernatant was introduced into a column of CM-Sephadex C-25 (1 x 30 cm), equilibrated with 0.05 M ammonium acetate, pH 4.6. After washing with the initial buffer, elution was performed with a gradient formed by passing 0.2 M ammonium acetate, pH 9.0, through a mixing flask containing 200 ml of the starting buffer. The PRL fraction was finally purified on a Sephadex G-100 column (2.64 x 47 cm). equilibrated with 0.05 M ammonium acetate, pH 6.8. Chetnicnl
and
physicochemical
characterization.
The purified PRL was examined by slab gel electrophoresis at pH 4.3 and 7.5 in 7.5% polyacrylamide gel (Grnstein, 1964) stained with 0.25% Coomassie brilliant blue R 250 in methanol-acetic acid-water (5: 1:5, v/v) (Meyer and Lamberts, 1965) and by gel isoelectric focusing in 5% polyacrylamide gel at 2% ampholine (pH 3.5-10 and pH 7-11) (Wrigley, 1971), stained with 0.04% Coomassie brilliant blue G 250 in 3.5% HCIO, (Reisner et a/.. 1975). The isoelectric point was estimated by gel isoelectric focusing with amphoiine (pH 7- 11). Protein samples containing PI-marker proteins (Oriental Yeast Co.) were focused in duplicate disc gels. One of the gels was stained and the other was sliced in 0.5 cm segments. The pH of each slice was measured after agitation in i ml of distilled water for 2 hr. The molecular weight was estimated by sodium dodecyl sulfate (SDS) slab gel electrophoresis (Weber and Osborn. 1969) and by exclusion chromatography on Sephadex G-100 using 0.1 1M Tris-HCl, pH 8.2. Egg white albumin, chymotrypsinogen, and lysozyme (Boehringer-Mannheim), and molecular weight markers (14,300-71,500) (BDH Chemicals) were employed as reference standards. Circular dichroism spectra were taken in a Gary model 60 spectropolarimeter, equipped with a Model 6002 circular dicbroism attachment in 0.1 M Tris-HCI, pH 8.2. The content of a-helix was estimated as described by Bewley et al. (1969).
PROLACTIN
447
An absorptivity value of the salmon PRL. pmc ,cm,esOnmwasdetermined to be 0.306 based on weight assuming a 15% moisture content. Protein concentrations were determined from absorption spectra after correction for light scattering as described by Beaven and Holiday (1952). For amino acid analysis, the protein was hydrolyzed for 22 hr at 110” with constant boiling HCl. The analysis was carried out with an I.323 automated amino acid analyzer (Type 4400). NH,-Termina? analysis was performed by the dansyl method (Gray, 1967) and COOH-terminal residue was determined by amino acid analysis of hydrazinolysate of performic acid oxidized PRL: according to Akabori et al. (1956). The amino acid sequence of the intact hormone was determined by the fluorescein-isothiocyanate as described previously (Muramoto et al., 1978). The fluorescein-thiohydantoin amino acids were identified by thin-layer chromatography on polyamide sheets. For further determination of the amino acid sequence, the protein was treated with cyanogen bromide, followed by performic acid oxidation. The resulting peptides were fractionated by gel filtration GII Sephadex G-50 in 0.1 N acetic acid and Sephadex G-75 in 0.1 N acetic acid, by droplet countercurrent chromatography on DCC-A (Tokyorika Co.) with !Ibutanol:pyridine:0.6 Iw ammonium acetate (pH 6 8) (5:3: 10, v/v), and high voltage paper electrophoresis in formic acid-acetic acid, pH 1.9 at 2000 V. Each fragment was digested by trypsin, chymotrypsin. and thermolysin. and subjected to high-performance liquid chromatography on reverse phase column C,, with 10 n&’ ammonium acetate (pH 4.0) and isopropanal. The amino acid sequence of these peptides were determined by the dansyl Edman procedures (Bruton and Hartley, 1970). Biologicai cizaracterizarion. Sodium-retaining activity of the salmon PRL was examined using intact tilapia and rainbow trout. The assay protocol for tilapia. Sarotkerodon mossambicus was the same as described by Clarke (1973). except that smaller fish weighing 1.5-3 g were used in the present study. They were acclimated to seawater (24 i- 1”) for more than 3 weeks before use. Ovine PRL (NIAMDD o-PRL-i3) was used as a reference standard. The fish were pretreated with 2 ,&g of ovine PRL for 3 days and subsequently given intraperitonial injections of saline (0.9% NaCI) or PRLs for 2 days. They were sacrificed 24 hr after the last injection. Blood samples were taken from the caudal artery following the method described by Jozuka and Adachi (1979). Similarly. assays v.ere carried using juvenile rainbow trout, S&no gnivd~eri, weighing 5-10 g. They were placed in 33% seawater for 3 days and then transfered to 50% seawater. Those fish acclimated to 50% seawaler for more than 7 days were injected intraperitoneally with stirnon or ovine PRL, and sacrificed 24 hr after the injection. Aiiquots
448
KAWAUCHI
of plasma (1 ~1 each) were diluted to 1 ml with deionized water and Na concentration was determined by atomic absorption spectrophotometer (Hitachi 208 or 180). Data are presented as means t SE. Multiple nonindependent comparison (Dunnett’s procedure) was applied to compare the means with a control. Immunochemical characterization. Antisera were raised against the salmon PRL in rabbits. The antigen (1 mg) was dissolved in 0.9% NaCl (1 ml) and emulsified with complete Freund’s adjuvant (1 ml). Seven injections, 1 mg each, were given subcutaneously into the foot pads and the back at intervals of 3 weeks. They were bled 2 weeks after the last injection, and the sera were lyophilized. The sera were tested by Guchterlony technique (Ouchterlony, 1953). Cross reactions of the antisera with ovine and whale PRLs (Kawauchi and Tsubokawa, 1979) and pituitary extract of tilapia were tested by agar diffusion techniques. An immunoglobulin (IgG) fraction was prepared from the antisera by ammonium sulfate fractionation. The antisalmon PRL rabbit IgG was used for immunocytochemical identification of PRL secreting cells in the chum salmon pituitary by peroxidase-antiperoxidase method (Sternberger et al., 1970). Details of this procedure will be described elsewhere (Naito et al., in press).
RESULTS
Chemical and physicochemical properties. The acid acetone extract of the salmon pituitary was subjected to gel tiltration on Sephadex G-25 to separate smaller peptides, such as endorphins and melanotropins from larger proteins as described previously (Kawauchi et al., 1980~). The unretarded fraction contained a large amount of NH,terminal peptide of salmon proopiocortins, which were removed by isoelectric precipitation (Kawauchi et al., 1981). As shown in Fig. 1, the supernatant consisted mainly of prolactin. When subjected to gel filtration on Sephadex G-100, the salmon PRL emerged as a single symmetrical peak with VJV, = 2.0. A yield of 50 mg was obtained from 50 g (wet weight) of the pituitaries. Polyacrylamide gel electrophoresis at pH 4.3 revealed only one intensely stained band, whereas no band was detected at pH 7.5. The isoelectric focusing of the salmon PRL exhibited a single band at pH 3.5% 10, and one intensely stained major band with a
ET AL.
second faint band running immediately behind it at pH 7- 11 (Fig. 2). The isoelectric point of the main band was estimated to be 10.3 (Fig. 3). The molecular weight of the salmon PRL was determined to be 22,300 by SDS polyacrylamide gel electrophoresis and 23,400 by exclusion chromatography on Sephadex G-100 (Fig. 4). The amino acid composition of the salmon PRL was calculated on the basis of a molecular weight of 22,300. As shown in Table 1, the total number of the residues by this calculation was 194, which is comparable to that in ovine PRL but exceeds tilapia PRL by 20 residues. A relatively high content of basic amino acids and low content of Glu are consistent with the high value of the isoelectric point. It is to be noted that the salmon PRL as well as tilapia PRL consisted of 4 half-cystine residues, which is in contrast with 6 residues in mammalian PRLs. As with tilapia PRL, relatively low content of tryptophan and tyrosine made the extinction coefficient low, 0.306. The high content of serine and methionine may be characteristic of salmon PRL. Isoleucine was identified as a sole amino-terminal residue by the dansyl method. The NH,-terminal sequence as determined by analysis of the intact hormone was H-Ile - Gly - Leu - Ser - Asp Leu - Met - Glu - Arg - Ala - . Hydrazinolysis of the intact hormone gave no amino acid, whereas that of the performic acid oxidized hormone yielded cysteic acid. Thus the COOH-terminal residue turned out to be half-cystine as are mammalian and tilapia PRLs. The salmon PRL gave 10 peptide fragments by cyanogen bromide cleavage and performic acid oxidation after fractionation using gel filtration, droplet countercurrent chromatography, and high voltage paper electrophoresis. The amino acid composition of these peptides is summarized in Table 2 and the total residues are consistent with that of the intact hormone. By
CHUM
SALMON
449
PROLACTIN
-------_-------
O*fq,~,O
_jt_
~X'~
QJN---..-
ACETATE
FIG. 1. Ion-exchange chromatography of the salmon PI& fraction on CM-Sephadex C-25. Initially, acid acetone extract of the salmon pituitary (50 g) was chromatographed on Sephadex G-25. The first fraction (390 mg) was subjected to isoelectric precipitation to remove a predominant acidic peptides: such as amino-terminal peptide of proopiocotrins (Kawaucbi et al., 1981). The supernatant was introduced to a column of CM-Sephadex C-25 (1 x 30 cm). Elution was performed with a gradient formed by passing 0.2 M ammonium acetate, pH 9.0, through a mixing flask containing 200 ml of the starting buffer at 3 ml/l0 minifraction.
analyzing amino acid sequence of the fragments as well as the intact hormone, CB-6 was identified to be the NH,-terminal peptide, followed by CB-5. It is evident that the salmon PRL lacks the NH,-terminal disule loop of the mammalian PRLs. CB-3 was assigned to the COOH-terminal peptide, due to the presence of half-cystine residue and absence of methionine at the carboxyl-terminal. CR-2 exhibited significant sequence homology with the COOHterminal portion of mammalian PRLs and GHs, especially of human PRL (172- 192). The partial amino acid sequence is aligned with the structures of mammalian PRLs and
GHs in Fig. 5. etails of studies on the mary structure will be described elsew The circular dichroism spectra are s in Fig. 6. The salmon negative band at 221 and which are characteristic of an a-helix. a-helix content was about 50% to nearest 5%. The spectrum in t side chain absorption exhibite peak at 291 nm with a shoulder around nm, a crossover point at 276 nm weak, negative shoulders near 267 nm. Biological
retaining
properties.
activity
The
of the salmon
s
pri-
the
300 two
261
450
KAWAUCHI
FIG. 2. Gel electrophoretic patterns of the purified salmon PRL. (A) Slab gel electrophoresis at pH 4.3; 50 pg on 7.5% gel stained with Coomassie brilliant blue R 250. (B) Gel isoelectric focusing at 2% ampholine (pH 3.5- 10) and (pH 7- 11); 50 pg each on 5% polyacrylamide stained with Coomassie brilliant blue G 250.
shown in Table 3. When tested in tilapia, the salmon PRL was inactive even at a dose of 20 pg/g, although the plasma sodium increased significantly after ovine PRL injection at 20 pgig. When injected into rainbow
ET AL.
0
0.4
0.6 MOBILITY
0.8
a
Lysozyme(l4600)
0.6 -
Salmon
r ; z % 0 .4-
PRL(22300) 0 = 2.0 f
(42900) 2 .0-
Salmon PRL(Z3400) Chymotrypsinogen (25000)
\
O-
1.5 4.0
4.5
5.0
Log nw
A
B
4. Determination of the molecular weight of the salmon PRL. (A) SDS gel electrophorcsis using molecular weight markers (14,300-71,500) obtained from BDH Chemicals (England). (B) Exclusion chromatography on Sephadex G-100 in 0.1 M Tris-HCl, pH 8.2. FIG.
, 1.0
FIG. 3. Estimation of the isoelectric point of the salmon PRL. The salmon PRL was focused in duplicate disc gel with ampholine (pH 7- 11). One of the gels was sliced in 0.5~cm segments for pH measurement, and the other was stained with Coomassie brilliant blue G 250. The pZ marker proteins consisted of cytochrome c from horse and the acetylated cytochromec; pZ, 6.4,8.3,9.7,and 10.6(OrientalYeastCo.).
2.5
0.s-
0.2
CHUM SALMON TABLE 1 AMINO Aclr, COMPOSITION OF SALMON PRL COMPARED WITH TILAPIA AND OVINE PRLs Amino acid
Salmonm PRL
Tilapia* PRL
Asp Thr Ser ml Pro Cl)’ Ala Cysi? Val Met Ble Leu TYr Phe Trp His LYS Arg
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 6.6 i3 .O 12.4
16.4 9.4 21.6 17.4 10.8 8.1 9.6 4.2 6.8 5.4 9.3 24.5 3.0 4.7 1.0 4.9 8.9 6.8
Ovine< PRL 22 9 15 22 11 11 9 6 10 7 11 23 7 6 2 8 9 11
PROLACTIN
45i
chum salmon pituitary with anti-salmon PRL rabbit hgGI showed strong cross reaction only in follicle forming cells of the rostral pars distalis at a dilution of 114000 (Figs, 8 and 9). These cells correspond with the PRL cells of chum salmon identified with light and electron microscopy (Nagahama, 1973): and distinguished from the GEL cells in the proximal pars distalis. No specific reaction was observed between the antibody and the GIL cells at any concentration tested. BISCUSSI0N
During the course of our studies on characterization of proopio~ort~~-~e~~te~ hormones from chum salmon pituitaries, prolactin was isolated as one of the predominant components from aci extract of the pituitary. Extraction by acid Total 193.4 172.8 199 acetone has been employed for isolation of mammalian PRLs as well as ACTHs and 0 Values were calculated on the basis of the molecular weight of 22,300. Threonine and serine values P-LPHs (Cole and Li, 1955) and proved to were corrected for destraction. Tryptophan was be an excellent procedure for obtaining determined by spectrophotometric method (Donovan, highly purified whale PRL and GE (Ka1969) and by methane sulfonic acid hydrolysis. wauchi and Tsubokawa, 1979; Tsubokawa b Taken from Farmer et al. (1977). et al., 1980). In contrast, fish irri3ve c Taken from Li et al. (1970). ctior; been prepared exclusively by at neutral or alkaline conditions. In such conditions, however, several undesirable trout acclimated to 50% seawater, both salmon and ovine PRLs caused significant side reactions may take place: enzymatic increase in plasma sodium after 24 hr, and 1 degradation will occur at neutral ~14, as ILg/g of the salmon PRL gave a similar re- well as deamidation and aggregation at sponse with 12.5 @gig of ovine PRL. alkaline pM. In addition, the chum salmon lmmurzoclzemical properties. Figure 7 PRL was proved to be a strongly basic shows an Ouchterlony agar diffusion plate protein and may not be extractable effiwith anti-salmon PRL rabbit serum in the ciently in alkahne condition. center well and ovine and whale PRLs and Most of the mammalian P the pituitary extract of tilapia in the tions have revealed several bands on pslyperipheral wells. A single strong precipitin acrylamide gel electrophoresis, and some of line was obtained between the antiserum the bands are assumed to be deamidated forms. The salmon PIPE exhibited only one and the salmon PRL. The pituitary extract of tilapia gave a reaction of partial identity electrophoretic band at pH 4.3 and in am3.5- IO) but a very faint and with the salmon PRL, but no line was observed between the antiserum and mammaslightiy acidic band in ampholine (pK lian PRLs. 7- 11). The minor component could be a Immunocytochemical staining of the deamidated form as are the cases Em mam-
452
KAWAUCHI
ET AL.
TABLE 2 AMINO ACID COMPOSITION OF CYANOGEN BROMIDE (CB) FRAGMENTS OBTAINED FROM SALMON PRL Amino acid Asp Thr Ser Glu Pro GUY Ala Cysl2” Val Met* Ile Leu Tv Phe His LYS Arg Trp Totald NHz-terminal residue
CB-1
CB-2
CB-3
9.6 2.2 10.6 8.3 6.2 3.3 4.0 1.5 2.0 + 4.8 13.9 1.0 0.4 1.9 6.1 2.9 +’
1.9 1.0 3.1
1.0
1.7 0.9 2.8 3.7
1.0
+ 2.9 3.8 0.8 3.0 1.0 2.0 0.9
(81)
24
5
33
28
7
Ser
A%
GUY
Glu
Ile
ND
1.0 0.9 0.7 2.0 0.8 + 0.6 1.6
CB-4
CB-5
CB-6
4.0 0.9 4.5 1.3 2.2 3.7
3.4 1.5 4.8 2.4 2.2
1.1
CB-7
CB-8
CB-9
CB-10
0.9
0.7
0.9 2.4
1.1
1.0
1.1 1.0 -
+ 4.0
+ 1.0 2.1
1.0 1.6 2.4 2.3
1.1 +
1.4 +
+
+
1.0 0.8 4 GUY
0.9 4
5
1
Val
Pro
Met
Note. The salmon PRL was treated with cyanogen bromide, followed by performic acid oxidation. The resulting peptides were fractionated on Sephadex G-50 and G-75 in 0.1 N acetic acid. The smaller peptides were separated by high voltage paper electrophoresis in formic acid-acetic acid, pH 1.9. The larger peptides were purified by droplet countercurrent chromatography with a solvent, n-butanol:pyridine:0.6 M ammonium acetate (pH 6.8) (5:3:10, v/v). a Determined after performic acid oxidation. b Homoserine + hormoserine lactone. r Detected by the uv absorption. d The values were obtained by sequence analysis except for CB-1.
malian PRLs. The isoelectric point of the salmon PRL (10.3) was notably higher than those of mammalian PRLs; p1 of ovine PRL is 5.6. An isoelectric point of the chum salmon PRL prepared by Idler et al. (1978) was reported to be 6.0. In contradiction with such a low isoelectric point, however, the protein was not absorbed on the anion exchanger of DEAE Bio Gel at pH 9.0. According to Farmer et al. (1975), tilapia PRL gave no electrophoretic band at pH 8.3. The same authors claimed later than a faintly stained band with an Rf of
band at pH 8.3 could not be a major but a minor or deamidated component. It seems more likely that the tilapia PRL as well as the salmon PRL is too basic to enter a gel at a slightly alkaline pH rather than that the protein is unstainable on the gel. The molecular weight of the salmon PRL was estimated to be 22,300, which is 3000 larger than that of tilapia PRL and similar to that of ovine PRL. Total amino acid residues were estimated to be 194, and amino acid analysis of ten cyanogen bromide fragments supports the estimation. The salmon PRL possessed four halfcystine residues or two disulfide bridges, as in the case of tilapia PRL. It has been speculated that the amino-terminal disulfide
Asp-Thr-Phe
0-Gti:
Glu-Phe-G1u-Arg-;h5r-Tyr-Ile
z!u-Phe-Glu-Glu-Ala-?&Ile
&$..His-Phe-Pro
Pro i-7 G,"-G,y-Gln-Arg--------------------------Leu-Le~-
( )-(
)-Arg-Phe-~8~-Glu-Ala-Ser
)-3:5--GlbGly-(
A?~-we-cooti
l;?;-Phe-COON
)-Ser
COOH
( )-Ser-(
)-Ile-His-a~~-Asn-Asn
coot4
coon
)-(
Ile-(
)--Ile-~:~-Aso-Am-Asn
Ala-Thr-Lys-Met-F&Pro-Gin-Thr
)-(
I?@-(
M',:-------------------------------------------------------------------------------------
NH2-Phe-Pro-Thr
NH,-Ala-Phe-Pro-Ala
FIG. 5. Partial amino acid sequence of the salmon PRL in comparison with mammalian PRLs and growlh hormones (GHs). The amino acid sequence of the salmon PKI. had been determined up to 35 residues at the NH,terminal and 30 residues at COOH-terminal. To &kin maximum homology with the other PRLs and CIIIs, the NIIYterminal residue of the salmon PRL was aligned with the 13th residue of the manmnlian PI&s. Three half-cystine residues at C~OF~-t~rrn~ll~i region were aligned in homologous positions to those of the mammalian hormones. The amino acid scqtlences are taken from ovine PRL (Li et al., 1970), human PKL (Shomc and Parlow, 15X77), ovine 611 (Li CT rrl., 1973); and human GH (Li, IY72).
Ser-Leu-5
Nt~,-Ceu-Pro-lle-~Cyz-Pro-Gly-Gly-Ala~-Ala-Arg-Cys-Gln
N~,-Thr-Pro-.Val-Cys-P:o-Asn-Gly-Pro-Gly-~~~-Cy~-Gl~-V~l~S~r NW,-Thr-Pro-.Val-Cys-P:o-Asn-Gly-Pro-Gly-~~~-Cy~-Gl~-V~l~S~r
S-PRL:
GH:
GH:
PRL:
Salmon
Ovine
PQL:
Hunan
HUIUII
PRL:
Ovine
z
F c: z! 2
z"
454
KAWAUCHI
ET
AL.
WAVELENGTH ( nm )
A
B
FIG. 6. Circular dichroism spectra of the salmon band absorption: (B) the side chain absorption.
PRL
bond in mammalian PRLs is absent in tilapia PRL, mainly because the aminoterminal tetrapeptide of the tilapia PRL revealed no half-cystine residue. In addition, selective reduction of the amino-terminal disulfide bond of ovine PRL increased the biological activity of the hormone, when assayed on permeability change in Gillichthys urinary bladder (Doneen et al.,
Fish
ACTIVITY
OF OVINE
Hormone
Tilapia
Saline o-PRL
s-PRL
Rainbow trout
Saline o-PRL
AND
SALMON
3 PRLs
Dose ~/-&g/g bw)
different
from
-
8 8 8 8 8 7 8
1
0.2 1 2.5 saline-injected
Number fish
5 10 20 0.5 5 20
5 12.5
* Significantly
IN TILAPIA
-
s-PRL
control
pH 8.2. (A) The amide
1979). As shown in the present studies, the maximum homology was found between the salmon PRL and mammalian PRLs and GHs, when the amino-terminal residue of salmon was aligned with the 13th residue of mammalian PRL. Three of the four halfcystine residues were identified at the carboxy1 terminal portion and aligned in the homologous positions with those of mam-
TABLE SODIUM-RETAINING
in 0.1 M Tris-HCl,
at P < 0.05.
of
AND
RAINBOW
TROUT
Plasma Na (mmoYliter) (mean i SE) 167 172 173 193 167 160 168
f IL * + t ? f
2.2 3.4 3.6* 6.0* 2.2 4.0 2.3
162.1 172.8 176.3 183.1 168.6 182.4 194.4
2 2 f + k -c -t-
4.7 5.4 2.7* 3.7* 3.2 2.9* 5.6*
CHUM SALMON
PROLACTlN
455
evidence for a common ancestral molecule for PRLs and GHs. Besides the similarity in the overall molecular features with the mammaIia~ hormones, the protein obtained in the present study was identified as by its immunological and biologica vities. Various hormone-secreting cells of the chum salmon pituitary have been identified by light and electron microscopy (Nagabama, 1973). The PRL cells in the chum salmon are arranged in the form of follic form the bulk of the rostra1 pars whereas GH cells are found ex~l~~ivel~ in the proximal pars distalis. In the present study, incubation of chum salmon pituFIG. 7. Immunodiffusion plate showing precipitin itaries with anti-salmon P reactions of salmon PRL, ovine PRL, whale PRL, and strong reaction only in the follicular pituitary extract of tilapia with the anti-salmon PRL cells, and no other cells in the pituitary rabbit serum in the center well. Photographed after 2 this reaction. In addition, pituitary extract hr of diffusion. of tilapia reacted with anti-salmon PRL in agar diffusion test, although precipitin lines malian hormones. Thus the salmon PRL hete definitely lacks one disulfide loop at the suggest immunological amino-terminal portion. However, it also partial identity) of these fish seems possible that the structure of the cular dichrois data, however, conformational similarity between the salprohormone of the salmon PRL is similar with that of mammalian PRL, and that the mon and tilapia P Ls: the side chain absorption spectra w e remarkably identical. processing of the prohormone could make of P entification the *‘phenotype” different. The resemblance of primary structure between mammalian PRLs and GHs the tilapia PRL antibodies bound strongly suggested that they have evolved RL and GH cells in two species of from a common ancestral molecule (Bewley s (Nagahama et al., 198I). and Li, 1971). In addition tilapia PRL as It has been well established im many telewell as some reptilian and amphibian PRLs ost species that PRL helps restore sodi have been shown to have some biological and immunochemical GH activities (Far- balance in fresh water by rnai~~a~~i~~ mer et nl., 1975; Nicoll and Licht, 1971; passive loss component throu a low level. On the ot Hayashida et nl., 1973). In our unpublished ment of fish in salt wa observation, the salmon PRL was imity to excrete sodium ions, t m~~olQgically active and parallel with of plasma sodium levels tilapia GII in two radioimmunoassay sys- an elevation (Clarke and Bern, 1980). Idler et al. (19778) tem employing rat GH antiserum (Hayashida, 1970) and snapping turtle GH an- have isolated salmon P retaining action in hyp tiserum (I-Iayashida et al., 197.5), although the activity was low. The sequence com- Poecilin. In the present s L was lo- IS times as active as ravine parison (Fig. 5) seems to provide further
456
KAWAUCHI
ET
AL.
FIG. 8. Sagittal section of Oncorhynchzrs beta pituitary stained with an anti-salmon PRL rabbit IgG at a concentration of 114000, counterstained with Meyer’s hematoxyline. RPD. rostra1 pars distalis; PPD, proximal pars distalis; PI, pars intermedia. x22. FIG. 9. Sagittal section of the rostra1 pars distalis of 0. keta pituitary stained with an anti-salmon PRL rabbit IgG at a concentration of 114000 counterstained with Meyer’s hematoxyline. Immunoreactivity is evident in PRL cells, whereas no reactivity is seen ACTH cells and GH cells. x264.
PRL in increasing plasma sodium of 50% seawater-acclimated rainbow trout, suggesting that its molecular structure is considerably specialized for an osmoregulatory role in salmonids. Similarly, tilapia PRL has been found to be approximately 100 times as active as ovine PRL in the tilapia sodium-retaining bioassay (Farmer et al., 1977). However, the salmon PRL was not active in tilapia assay, although ovine PRL was effective, indicating species specificity of fish PRLs. Absence of the tilapia sodium-retaining activity of the salmon PRL is in good accord with the original findings that pituitary extracts of salmonid
fishes were without effect in tilapia (Clarke, 1973). We have also tried to develop a homologous sodium-retaining assay using chum salmon fry adapted to seawater. However, salmon as well as ovine PRLs were without effect even though larger doses of the hormones were injected repeatedly. At any rate, sodium-retaining assay using rainbow trout as in the present study is far from satisfactory as a bioassay for fish PRL. Further studies are needed to increase accuracy and validate specificity of the assay in order to provide conclusive evidence for the physiological role of the salmon PRL.
CHUM
SALMON
ACKNOWLEDGMENTS We thank Hiroto Okamura and Ryutaro Kudo for their skilled technical assistance. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture to H. K. and T. H.. and also from the Japan Society for Promotion of Science to T. H.
REFERENCES Akabori, S., Ohno, K.. Ikenaka, T., Okada, Y., Hanafuss, H., Haruna, I.. Tsugita, A., Sugae, K., and Matsushima, T. (1956). Hydrazinolysis of peptides and proteins. II. Fundamental studies on the determination of the carboxyl ends of proteins. Bull. Chern. Sm. J~~purr 29, 507-518. Beaven, 6. H., and Holiday, E. R. (1952). Ultraviolet absorption spectra of proteins and amino acids. Atl~rn. Protein Chem. 7, 319-386. Bern. II. A. (1975). Prolactin and osmoregulation. Amer. Zool. 15, 937-948. Bewley. T. A., Brovetto-cruz, .I., and Li, C. H. (1969). Human pituitary growth hormone. Physicochemical investigations of the native and reducedalkylated protein. Biochemisrry 8, 4701-4708. Bewley, T. A., and Li, C. H. (1971). Sequence comparison of human pituitary growth hormone, human chorionic somatomammotropin and ovine pituitary lactogenic hormone. Experierriia 27, !368- 1371. Bruton, C. J.. and Hartley. B. S. (1970). Chemical studies on meihionyl-tRNA synthetase from Escherichicr coli. i. Mol. Bio/. 52, 1655 178. Clarke, W. C. (1973). Sodium-retaining bioassay of prolactin in the intact teleost Ti/apia mossambica acclimated to seawater. Gen. Conzp. .%ducvinol. 21, 498-512. Clarke. W. C.. and Bern, H. A. (1980). Comparative endocrinology of prolactin Irr “Hormonal Proteins and Peptides” (C. H. Li. ed.), Vol. 8, pp. iCt- 197. Academic Press, New York. Cole. R. D.. and Li. C. H. (1955). Studies on pituitary lactogenic hormone. XIV. A simplified procedure of isolation. J. Biol. Che/?l. 213, 197-201. Doneen: B. A., Bewley, T. A., and Li, C. H. (1979). Studies on prolactin. Selective reduction of the disulfide bonds of the ovine hormone. Biochemistry 18, 4851-4860. Donovan. .I. W. (1969) Ultraviolet absorption. 112 “Physical Principles and Techniques of Protein Chemistry” (Leach. S. J., ed.), Part A, pp. IOl- 170. Academic Press, New York. 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. Lv‘e SC?. 16, 1499 i.58. Farmer: S. W.. Papkoff: H.. Bewley. T. A..
PROLACTIN
457
Hayashida, T., Nishioka, R. S., Bern, II. A., and Li, C. H. (1977). Isolation and proper?ies of teieost proiactin. Getz. Camp. Endocrinoi. 31, M-71. Gray. W. R. (1967). Sequential degradation plus dansylation. In “Methods in Enzymology” (C. H. W. Hirs, ed.); Vol. II, pp. 469-475. Academic Press, New York. Hayashida. T. (1970). Immunoiogicai studies with rat pituitary growth laormone (RGH) II. Comparative immunochemical investigation of GH from repre sentatives of various vertebrate classes with monkey antiserum to RGH. Gen. Camp. Enciooiricii 15,432-452. Hayashida, T.. Licht. P., and Nicoll, C. S. (1973). Amphibian pituitary growth hormone and proiactin: Immunochemical relatedness to rat growth hormone. Science 182, 169- 171. Hayashida, T.. Farmer, S. W.. and Papkoff, H. ( 1975). Pituitary growth hormones. Further evidence for evolutionary conservatism based on immunochemical studies. Proc. Wnr. i\cati. Sci USA 72, 4322-4326. Idler, I), R., Shamsazzaman, K. M., and Burton, M. P. (1978). Isolation of prolactin from salmon pituitary. Gerr. Camp. Etzdocrinoi. 35, 409-438. Jozuka, K., and Adachi, II. (1979). Environmenta! physiology on the pH tolerance of teleost. II. Blood properties of medaka. Orr:irr.\ laf;pc.x. ex-posed to low pI5 environment. At~lrof. Zool. Jupun 52, 107-113. Kawauchi. H., and Tsubokawa, M. (1979). Isolation and characterization of fin whale prolactin. ir!t. .i. Pepride Proreirr Res. 13, 229-234. Kawauchi, II., Tsubokawa, M.. Kanerawa. A., and Kiiagawa, H. (1980a). Occurrence oftwo different endorphins in the salmon pituitary. Biocherrr. Biophys. Rrs. Cornmnn. 92, 127% 1288. Kawauchi. II.: Ada&i, Y., and Tsubokawa. M. (1980b). Occurrence of a new melanocyte stimulating hormone in the salmon pituitary glands. Biochem. Bi0ph.v~. Res. Ctinim~i2. 90, 1508- 1517. Kawauchi, H.. Adachi. Y.. and Ishizuka, B. (I%&). Isolation and structure of another /3-melanotropin from salmon pituitary glands. Int. J. Prplitic Protein Res. 16, 79982. Kawauchi, II.. Takahashi, A., and Abe. K. (1981). Gamma-melanotropin is not present in an 2% terminal peptide of salmon proopiocortin. 1111. /. Peptide Protein Res. 18, 223-727. Kawauchi, II., Takahashi, A., and Abe. Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pimitary glands. ArcA. Biochem. Biophyr. 213, 680-688. Li, C. H., Dixon, 3. S.. Lo. T-B.. Schmidt, K. D., and Pankov. Y. A. (1970). Studies on pituitary iactogenic hormone. XXX. The primary strucrcre cf
458
KAWAUCHI
the sheep hormone. Arch. Biochem. Biophys. 141, 705-737. Li, C. H. (1972). Hormones of the adenohypophysis. Proc. Amer. Phil. Sot. 116, 365-382. Li, C. H., Gordon, D., and Knorr, J. (1973). Primary structure of sheep pituitary growth hormone. Arch. Biochem. Biophys. 1.56, 493-508. Meyer, T. S., and Lamberts, B. L. (1965). Use of Coomassie brilliant blue R 250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide gel strips. Biochim. Biophys. Acta, 107, 144- 145. Muramoto, K., Kawauchi, H., and Tsuzimura, K. (1978). Sequence determination of peptide by the combined use of fluorescein isothiocyanate and phenylisothiocyanate. Agric. Biol. Chem. 42, 1559- 1563. Nagahama, Y. (1973). Histophysiologica! studies on the pituitary gland of some teleost fishes, with special reference to the classification of hormone-producing cells in adenohypophysis. Mem. Far. Fish. Hokkaido Univ. 21, l-63. Nagahama, Y., Olivereau, M., Farmer, S. W., Nishioka, R. S., and Bern, H. A. (1981). Immunocytochemical identification of the prolactinand growth hormone-secreting cells in the teleost pituitary with antisera to tilapia prolactin and growth hormone. Gen. Comp. Endocrinol. 44, 389-395. Naito, N., Takahashi, A.. Nakai, Y., Kawauchi, H., and Hirano, T. “Immunocytochemical Identification of the Prolactin-Secreting Cells in the Teleost Pituitary with Antiserum to Chum Salmon Prolactin.” Gen. Comp. Endocrinol., in press. Nicoll, C. S., and Licht, P. (1971). Evolutionary biology of prolactins and somatotropins. II. Electrophoretic comparison of tetrapod somatotropins. Gen. Comp. Endocrinol. 17, 490-507.
ET AL. Ng, T. B., Idler, D. R., and Bruton, M. (1980). A comparison of prolacting from a marine, an estuarine, and a freshwater teleost. Gen. Comp. Endocrinol. 42, 141- 146. Ornstein, L. (1964). Disk 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. Reisner, A. H., Nemes, P., and Bucholtz, B. (1975). Use of Coomassie brilliant blue G 250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels. Anal. Biochem. 64, 509-516. Shome, B., and Parlow, A. F. (1977). Human pituitary prolactin (PRL): the entire linear amino acid sequence. .I. Clin. Endocrirlol. Metab. 45, 1112-1115. Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., and Meyer, H. G. (1970). The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. .I. Histochem. Cytochem. 18, 315-333. Tsubokawa, M., Kawauchi, H., and Li, C. H. (1980). Isolation and partial characterization of growth hormone from fin whale pituitary glands. J. Biochem. 88, 1407- 1412. Weber, K., and Osborn, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. Wrigley, C. W. (1971). Gel electrofocusing. In “Methods in Enzymology” (W. B. Jakoby, ed.), Vol. 22, pp. 559-564. Academic Press, New York.