Int. J. Peptide Protein Res. 13, 1919,229-234 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

ISOLATION A N D CHARACTERIZATION OF FIN WHALE PROLACTIN* HIROSHI KAWAUCHI and MAKOTO TUBOKAWA

Kitasato University, School of Fisheries Sciences, Sanriku-cho. Iwate, Japan

Received 30 May, accepted for publication 14 June 1978 A highly purified prolactin preparation has been obtained from fin whale pituitaries by extraction with acid acetone, salt precipitation, isoelectric fractionation, and exclusion chromatography on Sephadex G-1 00 and ionexchange chromatography on DEAE-cellulose. Fin whale prolactin was isolated in a yield of 250mglkg wet weight tissue. It was found to have a molecular weight (SDS disc gel electrophoresis) o f 23,600 daltons and an a-helix content (circular dichroism) of 50%. The amino acid composition and circular dichroism spectra were very similar to those of porcine prolactin. The partial amino acid sequence has been determined by the method of fluorescein-isothiocyanate. Fin whale prolactin was found to be 80% as potent as ovine prolactin with regard to pigeon crop-sac assay.

Key words: characterization;isolation; primary structure;whale prolactin.

The presence of prolactin has been reported in the pituitary glands of various vertebrates. Highly purified preparations of the hormone have been obtained from ovine, bovine and porcine, and complete amino acid sequences of them have been elucidated (Li et aZ., 1970; Wallis, 1974; Li, 1976). Prolactin consists of a single polypeptide chain of 199 amino acid residues containing three disulfide bridges. The comparison of amino acid sequences indicates that these prolactins are highly homologous. This communication reports the isolation and characterization of fin whale prolactin, including partial amino acid sequence. EXPERIMENTAL PROCEDURES Isolation

The fin whale pituitaries were generously supplied by Nihonsuisan Co. (Tokyo). The

* Presented

at the “Free Communication” of the International Symposium on Proteins, Taipei, Taiwan,March 6-9,1971.

session

0367-8377/79/020229-06

glands were collected within 5 h after the capture in the South Pacific Ocean and stored at - 30”. The whale pituitary prolactin was initially extracted as described by Cole & Li (1955). The crude prolactin fraction was further purified by NaCl precipitation, isoelectric fractionation, gel filtration of Sephadex G-100 and ionexchange chromatography on DEAEcellulose. Chemical characterization

All fractions were examined by polyacrylamide disc gel electrophoresis at pH7.5 in 7.5% gel (Omstein, 1964) stained with Amido Schwarz. The purified hormone was also tested by gel isoelectrofocusing at a concentration of 2% ampholine (pH3.5-10) in 5% gel (Wrigley, 1971), stained with 0.04% CBB-G-250 in 3.5% HCIO., (Reisner et (11.. 1975). The molecular weight of the prolactin was estimated by SDS disc electrophoresis (Weber & Osborn, 1969), with pepsin, ovine prolactin and Iysozyme as standard proteins.

$02.00/0 0 1979 Munksgaard, Copenhagen

229

H. KAWAUCHI and M. TUBOKAWA

Circular dichroism spectra were taken in a Cary Model 60 spectropolarimeter equipped with a Model 6002 circular dichroism attachment in 0.1 M Tris-C1, pH8.2 (Bewley et al., 1972). Protein concentration was determined spectrophotometrically using the relation =9.09 as previously reported for ovine prolactin (Li el ul., 1970).

EiZ

Primary structure

HCl. The yield of the crude prolactin was 400mg. The fraction was further purified on a column of Sephadex G-100 (3 x 45 cm), equilibrated with 0.1 M Tris-C1, pH8.2 as shown in Fig. 1A. Fraction C was rechromatographed on the same column as shown in Fig. 1B.The final purification was achieved in a column of DEAE-cellulose (1.8 x 24 cm), equilibrated with 0.03 M ammonium carbonate, pH 9 .O, Elution was performed with a gradient (formed by passing 0.3 M ammonium carbonate, pH9.0, through a 300-ml mixing flask containing the starting buffer of 0.03 M ammonium carbonate, pH9.0). The elution pattern is shown in Fig. 2. The yield of purified prolactin was 250mglkg wet weight tissue.

Amino acid analyses were performed by the method of Spackman et al. (1958) in a Hitachi automatic analyzer Type 034. The protein was treated with cyanogen bromide, followed by performic acid oxidation. The resulting peptides were fractionated by exclusion chromatography on Sephadex and ion-exchange chromatography with CM cellulose. Amino - and carboxyl -terminal residues were determined by the Edman (Gray & Hartley, 1963; Woods & Wang, 1967) and carboxypeptidase (Ambler, 1972) methods, respectively. Sequence analysis was performed by the fluorescein-isothiocyanate method (Maeda & Kawauchi, 1968; Muramoto et QI., 1978), directly identifying fluorescein- E thiohydantoin amino acid on polyamide thin- c layer plates (Muramoto et al., 1975). Y)

N

Bioassay

The biological activity of highly purified fin whale prolactin was measured by the local pigeon crop-sac assay with ovine prolactin (NIH-P-S-8) employed as standard (Tanabe et ul., 1954). 08

10

12

Isolation and purification

700g of fin whale pituitaries (45 glands) was extracted with acid acetone according to the method of Cole & Li (1955). The yield of acetone precipitate was 14 g. The material was dissolved in water and the pH adjusted to 3.0. Sodium chloride was added to a concentration of 6% NaCl saturation. The NaCl precipitate (4.2g) was dissolved in water at pH 8.5. The resulting precipitate was removed and the pH of supernatant was successively lowered to values of 7.5, 7.0, 6.5, 6.0, and 5.6 with 0.1 N 230

14

16

ia

M

zz

w vo

RESULTS

FIGURE 1 Gel filtration of fin whale prolactin on Sephadex G-100 column (3 X 45 cm) with 0.1 M Tris-Cl, pH 8.2. A, crude prolactin (100 rng); B, Fraction C (10 rng).

Gel electrophoresis

Disc gel electrophoretic pattern and gel electrofocusing pattern of fin whale prolactin are present in Fig. 3. The purified prolactin had an additional band, which is characteristic and believed to be the deamidated form of prolactin and not an impurity.

FIN WHALE PROLACTIN

The molecular weight of tin whale prolactin was determined to be 23,600 by SDS polyacrylamide gel electrophoresis using pepsin, ovine prolactin and lysozyme empioyed as standard as shown in Fig. 4.

Circular dichroism

Circular dichroism spectrum of tin whale prolactin below 250nm at pH8.2 is shown in Fig. 5A. The hormone showed two negative bands characteristic of a-helical content and was estimated to be about 50%. CD spectrum in the region of side chain absorption at pH 8.2 is presented in Fig. 5B. The protein showed a very weak positive band with a maximum around 298 nm, and a negative maxima at 290, 281, and 268nm with gradually increasing negative ellipticity. Biological activity

The biological activity of fin whale prolactin was determined by local pigeon crop-sac assay. The prolactin exhibited a potency of 22.7k2.68 IU/mg relative to a standard ovine prolactin (NIH-P-S-8,28 IU/mg). Amino acid sequence

20

40

60

80

100

120

110

160

Tube no.

FIGURE 2 DEAEcellulose chromatography of fin whale prolactin rechromatographed on Sephadex G-100 ( F i g . 1B) by gradient elution as shown, column size (1.8 x 24 cm), flow rate: 2.8 m1/7 midtube.

Amino acid composition of fin whale prolactin, calculated on the basis of a molecular weight of 23,600 daltons, is shown in Table 1. Aminoand carboxyl-terminal residues were identified to be leucine and half-cystine, respectively. Cyanogen bromide fragmentation of the hormone (50mg) gave two fractions, CB-I and CB-I1 on Sephadex G-75 and 0.1 N AcOH. CB-I (25mg) was oxidized by performic acid, and separated into two fractions, 0-CB-IA, and O-CB-IB on Sephadex G-75 in 10%formic acid. CB-I1 (1 5 mg) was fractionated on CM-cellulose chromatography into three fractions, CB-I1A, CB-IIB, CB-IIC. Partial amino acid sequences

f

+ A

B

C

FIGURE 3 A, Disc gel electrophoretic pattern of fin whale prolactin (Fraction C) at pH 7.5in 7.5% gel stained with Amido Schwartz. B, Gel electrofocusing pattern of Fraction C and C, purified fm whale prolactin at 2% ampholine (pH3.5-10) in 5% gel stained with 0.04%CBBG250 in 3.5% HCIO,

.

23 I

H. KAWAUCHI and M. TUBOKAWA

5 P

B

2 -

4" :

Popsin

\ (in Whale prohctin

3 -

o w e prdactm lysozyrne

2 -

0

0

I

1

as

1

1

I

I

FIGURE 4 Determination of molecular weight by SDS gel electrophoresis of fin whale prolactin. The standard proteins were

FIGURE 5 Circular dichroism spectrum A, below 250 nm and B, side chain absorption for fin whale prolactin in 0.1M TrisC1, pH 8.2. nm

of these peptides could be determined up to 20- Minor heterogeneity was observed by gel 25 residues using 20-30nmol of peptide as electrofocusing. Further purified prolactin from DEAE-cellulose chromatography gave only a shown in Fig. 6. faint band of the deamidated form. Our preparation of fin whale prolactin was about DISCUSSION 80% as potent as ovine prolactin in the local This is the first publication on the isolation and pigeon crop-sac assay. characterization of whale prolactin. Fin whale Overall conformation of fin whale prolactin prolactin was isolated in a yield of 250mglkg observed by circular dichroism was very wet weight tissue by the method of Cole & Li. similar to other mammalian prolactin, The prolactin obtained from Sephadex G-100 especially porcine hormone (Bewley & Li, was homogeneous by SDS gel, and polyacryl- 1975). This observation was further supported amide gel electrophoresis at pH 7.5 except for by the amino acid composition and partial an additional band which is believed to be a amino acid sequence. The hormone contained deamidated form (Cheever & Lewis, 1969). four methionine residues and three disulfide 232

FIN WHALE PROLACTIN wP(o-cp-II-c 1

:

PP

:

(5) (101 (15) (20) (251 H-Leu-Pr~-Ilo-CyS-Pro-Ser-Gly-Ala-Val-A~~-Cy~~-Gln-Val-Ser-~~-Arq-Asp-Le~-Ph~-A~~-Arq-Al~-Val-lle-~u-

H-Leu-Pra-I1e-Cy~-Pro-SeT-Gly-Ala-Val-A~~-Cys-Gl~-Val-Ser-~~-Arq-Asp-Leu-Phe-Asp-Arg-Al~-Val-lle-L~~5 10 15 20 25 (351

(30)

-Ser-HiS-Tyr-lle-H1S-Asn-Leu-Ser-Ser-Glu-Met-OH -Ser-His-Tyl-Ile-HIS-Am-Leu-Ser-Ser-Glu-Met-

WP 10-CH-I-A1

:

PP

:

3n

35

(51

(101

lt-Phe-Asn-~lu-Phe-As~-Lys-Rrq-Tyr-Al~-Gl~-Gly-Arq-

-Met-OH

-Phe-Asn-Gle-Phe-Asp-l.yS-Arq-Tyr-Ala-Gln-G1y-Arq-Gly-Phe40 45 50

-Arq-Gly-Met105

(51 (10) (15) (20) (25) H-Gln-GlU-Al~-P~O-A~p-Ala-Ile-~u-Se~-A~g-Al~-Ile-Glu-Ile-Glu-Gl~-Gln-Asn-Ly~-A~q-Leu-Leu-Glu-Gly-~t-OH

WP ICB-11-A)

:

PP

:

WP(CR-11-0)

:

(51 (101 (151 (20) (25) H-Glu-Ly5-Ile-Val-G1y-Gln-Val-His-P~~~Gly-val-Ly~-Glu-A~n-Glu-Val-Ty~-Ser-V~l-T~p-Se~-Gly-Leu-P~a-Se~-

PP

:

-Glu-Lys-Ile-Val-Gly-Gln-Val-His-P~~-Gly-Ile-Lys-Glu-A~n-Glu-val-~r-Ser-Val-Trp-S~r-Gl~-Leu-P~a-Se~-

- G l ~ - G 1 U - A l ~ - P ~ O - A ~ p - A l ~ - l l ~ - L e U - S e T - R r g u - G l u - G l y - ~ ~ t iin 115 120 125 130

135

140

150

145

155

-Leu-Gln-Met-OH -Leu-Gln-Met-

WP(0-CB-I-B)

:

PP

:

(51 (10) (151 (20) H-Ala-ASp-G1U-ASp-Th~-A~q-Leu-Phe-Ala-Phe-~r-Asn-~~-~~~-Hi~~~-~u-A~q-A~q-A~p-Ser-His-Lys-

-Ala-Asp-Glu-Asp-Thr-A~~-~u-Phe-Ala-Phe-~~-Asn-~u-~u-Hi~-cys-~u-Arq-Arq-A~p-Se~-~i~-Lys-Ile-A~p160

165

170 -Cys-Arg

185

175

180

Ile-Ile-Tyr-Aax-Ser-Am-Cya-OH

190

195

FIGURE 6 Amino acid sequence of peptides obtained from cyanogen bromide cleavage and performic acid oxidation of fin whale prolactin (WP) as compared with the structure of porcine prolactin (PP) (Li, 1976).

bridges, and cleavage by cyanogen bromide and performic acid oxidation yielded five peptide fragments, as is the case with the porcine hormone (Li, 1976). The amino acid sequence of these peptides so far studied was almost identical to those of porcine prolactin. The differences found were at Gln-122 and Val-141. The complete sequence analysis is in progress in our laboratory.

REFERENCES Ambler, R.P (1972) in Methods in Enzymology (C.H.W. Hirs & S.N. Timasheff, eds). vol. 25,pp. 262-271,Academic Press, New York Bewley, T.A. & Li, C.H. (1975) Arch. Biochem. Biophys. 167,80-90 Bewley, T.A., Kawauchi, H. & Li, C.H. (1972)Biochemistry 1 1,41 79-4 187 Cheever, E.U. & Lewis, U.J. (1969)Endocrinology

85,465-473

ACKNOWLEDGEMENTS We thank Dr. Choh Hao Li for his advice and encouragement and Dave Chung for the critical reading of this manuscript. We are grateful to Koji Muramoto for his assistance in the sequence analysis and Dr. Kaoru Komoto for the bioassay data. This study was supported in part of the Naito Research Grant for 1977.

Cole, R.D. & Li, C.H. (1955) J. Biol. Chem. 213, 179-20 1 Gray, W.R. & Hartley, B.S. (1963)Biochem. J. 89,

59P Li,C.H. (1976)Int.J. Pept. h o t . Res. 8,205-224 Li, C.H., Dixon, J.S., Lo, T.B., Schmidt, K.D. & Pankov, Y .A. (1970) Arch. Biochem Biophys.

141,705-737 Maeda, H. & Kawauchi, H. (1968)Biochem. Biophys. Res. Commun. 31,188-192

233

TABLE 1

Muramoto, K., Kawauchi, H. & Tuzimura, K., (1975) Agnk Biol. Chem. 39,2241-2242 Muramoto, K., Kawauchi, H. & Tuzimura, K. (1978) Amino acid Fin whale Ovinea Bovineb Porcine= Agric. Biol. Chem. 42,1559-1563 Ornstein, L. (1964) Ann. N.Y. Acad. Sci. 121, 3219 9 9 349 9.3 LY s 8 7 9 8.2 Reisner, A.H., Nemes, P. & Bacholtz, C. (1975) Anal. His 11 11 13 12.1 Biochem, 64,506-516 Arg 22 22 22 Spackman, D.H., Stein, W.H. & Moore, S . (1958) 21.1 ASP 9 9 5 Anal. Chem. 30,1190-1206 5.8 Thr 16.3 15 15 16 Tanabe, Y., Shoda, Y. & Sakai, Y. (1954) Bull. Natl. Ser 22 22 24 25.5 Glu Inst. Agr. Sci. Series G, No. 9,57-65 11 11 7 Pro 8.O Wallis, M.(1974) FEBS Letters 44,205 -208 11 11 8 89 Weber, K. & Osborn. M. (1969) J. Biol. Chem. 244, G~Y 9 10 10 12.4 Ala 4406-4412 Woods, K.R. & Wang, K.T. (1967) Biochim. Biophys. 6 6 6 5.8d Cysl2 10 9 11 13.1 Val Acta 133,369-370 7 7 4 4.0 Met Wrigley, C.W. (1971) in Methods in Enzymology, 11 11 15 11.6 Ile (W.B. Jacob, ed.), vol. 22, pp. 559-564, Academic 23 23 26 24.0 Leu Press, New York 7 8 7 6.8 Tyr 6 6 6 6.0 Phe Address: 2 2 2 2e TIP Hiroshi Kawauchi a Taken from Li et al. (1970). Kitasato University b Taken from Wallis (1974). School of Fisheries Sciences C Taken from Li (1976). Sanriku+ho d Determined after performic acid oxidation. lwate 022-01 Determined by spectrophotometric method. Japan Amino acid composition of fin whale prolactin

234