Studies on prolactin 48: Isolation and properties of the hormone from horse pituitary glands

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 220, No. 1, January, pp. 208-213, 1983

Studies on Prolactin 48: Isolation and Properties the Hormone from Horse Pituitary Glands CHOH Hormone

Research

Laboratory, Received

HA0 1088 HSW,

May

LI’

AND

University

DAVID of California,

27, 1982, and in revised

form

of

CHUNG San Francisco, August

Califwnia

g&&3

24, 1982

Isolation of prolactin from equine pituitary glands has been described. It has a potency of 42 IU/mg in the pigeon crop-sac test and consists of 199 amino acids. The hormone has only four half-cystine residues in contrast to other mammalian prolactins which have six residues. From NHa-terminal sequence analysis and amino acid composition of cyanogen bromide fragments, the NHB-terminal disulfide loop is missing in the equine prolactin molecule. Circular dichroism spectra indicate that the a-helical content of equine prolactin appears to be lower (50%) than that found in the ovine hormone (65%).

chased from Burdick and Jackson Laboratories (Muskegon, Mich.) and TFA (redistilled) from Eastman Kodak. Carboxypeptidase Y was a commercial product (Pierce Chemical Co., Rockford, Ill.). HPLC was performed on a reverse-phase column (4.6 X 250 mm, VYDAC, 201 TP, C-6000, Cls, 300 A) from Alltech Associates (Deerfield, Ill.). The chromatography system consisted of the Laboratory Data Control (Riviera Beach, Fla.) gradient master, two constametric pumps (Constametric I and II G), mixer, and Spectromonitor III variable wavelength monitor. The absorbance at 210 nm was detected with a Heathkit Model SR-255-B (Benton Harbor, Mich.) recorder. Amino acid analysis was performed according to the method as described (11) in an automatic amino acid analyzer (Model 119C, Beckman Instruments). Proteins (ca. 0.1 mg) were hydrolyzed with constantboiling hydrochloric acid (500 ~1) in evacuated, flushed with Na, and sealed ampules for 20 h at 110°C or with methanesulfonic acid (12). NH,-terminal analysis was carried out by the dansyl procedure (13,14) and the dansyl-Edman method was employed for sequence analysis as previously described (13). Oxidation of ePRL with performic acid was performed as described (15). Reduction and alkylation with iodoacetamide were carried out according to the procedure of Graf et al. (16). SDS-gel electrophoresis was performed by the procedure of Swank and Munkres (17) with modifications as described (16). Electrophoresis on columns of polyacrylamide (7%) at pH 8.3 was carried out for 60 min with 3 mA per tube. CD spectra were obtained in a Cary Model 60 spec-

Since the discovery of lactogenic or prolactin activity in bovine pituitary extracts (l), the hormone has been isolated from glands of various species (2) and their amino acid sequences have also been determined [sheep (3), bovine (4, 5), porcine (6), rat (7, 8), and human (9, lo)]. It is a protein consisting of 199 amino acids with three disulfide bridges. We report here that equine PRL2 has only two disulfide bridges with the same number of amino acid residues. In the pigeon crop-sac assay, equine PRL has the same potency when compared with the ovine hormone. MATERIALS

AND

METHODS

Horse pituitary glands were purchased from Central Nebraska Packing Company (North Platte, Nebr.). Sephadex G-100 was obtained from Pharmacia (Piscataway, N. J.) and DEAE-cellulose (Cellex D) from Bio-Rad (Richmond, Calif.). Propanol-2 was pur-

‘Author to whom all correspondence should be addressed. ’ Abbreviations used: PRL, prolactin; ePRL, equine PRL; oPRL, ovine PRL, DEAE, diethylaminoethyl; TFA, trifluoroacetic acid; SDS, sodium dodecyl sulfate; Cam, carbamidomethylated; CD, circular dichroism; HOAc, acetic acid; PyrAc, pyridine acetate. 0003-9861/83/010208-OG$O3.00/0 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

208

EQUINE

209

PROLACTIN

060 OIM -1-l g ?! z 0 0

005M

OIM I

04-

02I Xl Tube

FIG. 1. Gel filtration of partially purified ePRL (300 mg) on Sephadex G-100 column (2.4 X 84 cm) in pH 8.2,O.Ol M NH,HCO,; 25 ml/h; 5 ml/tube. The content in tubes 60-75 contained ePRL.

tropolarimeter equipped with a Model 6002 CD attachment. The sample was dissolved in 0.1 M Tris-HCl buffer of pH 8.2. The concentration of the solution was determined spectrophotometrically using the relation .F” 1 cm,218 “In = 0.849. To cleave methionyl peptide bonds, ePRL (10 mg) was treated with cyanogen bromide in 70% (v/v) formic acid (18, 19). The reaction mixture was allowed to stand at 22°C in the dark for 24 h and then diluted lo-fold with distilled water and lyophilized. The dried product was dissolved in 0.1 M HOAc and submitted to gel filtration on a Sephadex G-50 column (1.5 X 52 cm) using the same solvent for elution. The CNBr fragments were further purified by electrophoresis on paper (Whatman 3MM) in the collidine-acetic acid buffer of pH 7.0 at 400 V for 2 h. Lactogenic activity was estimated in the pigeon crop-sac assay (20) as modified by Nicoll (21) using oPRL (3) as standard. RESULTS

Acid acetone extract of 300 g of whole horse pituitaries was obtained as described (22). The resulting acid acetone powder (12 g) was dissolved in distilled water (1200 ml) and adjusted to pH 3.0. The solution was brought to 6% saturation with respect to NaCl and allowed to stand at 4°C for 18 h. The precipitate3 was dissolved, dialyzed, and lyophilized. The lyophilized material (3.3 g) was dissolved in aqueous solution (300 ml) of pH 9.5. After standing overnight at 4°C the insoluble material was removed by centrifugation and the clear supernatant was adjusted to 3 The supernatant of P-endorphin (23-25).

was employed and other biologically

for

the isolation active peptides

I 40 number

I 50

FIG. 2. Chromatography of purified ePRL (116 mg) on DEAE-ion-exchange column (2.2 X 30 cm); pH 7.0, NH,OAc buffer; 10 ml/tube. The content in tubes 3540 contained ePRL.

pH 5.7 with 1 M HCl. The isoelectric precipitate was recovered by centrifugation, dissolved in aqueous solution of pH 8.0, and lyophilized (0.8 g). The partially purified ePRL (300 mg) was further purified by gel filtration on Sephadex G-100 in 0.01 M NH,HCO, of pH 8.5 as shown in Fig. 1. There were two main peaks4 and the more retarded material ( V,/ V. = 2.10) was identified to be purified ePRL (56 mg) by bioassay. The purified material (116 mg) was next submitted to chromatography on DEAE-cellulose column (Fig. 2); the content in the main peak was dialyzed, lyophilized, and yielded 23 mg highly purified ePRL. Table I presents the protocol of the isolation procedure. From 1 kg of whole pituitary glands, 130 mg of ePRL may be isolated to homogeneity. SDS-gel electrophoresis of ePRL gave a single band (data not shown). When compared with standard proteins of known molecular weight, the molecular weight of the hormone was estimated to be 22,500. In disc electrophoresis, ePRL migrated as a single component at pH 8.3 as shown in Fig. 3. HPLC of ePRL showed that the preparation behaved as a homogeneous protein (Fig. 4). The CD spectra of ePRL and oPRL (3) in the region dominated by the amide bond absorption are shown in Fig. 5. Both ‘The less retarded material (V,/V, = 1.70) had Phe as the NH,-terminal residue. Bioassay by the tibia test indicated that it had growth-promoting activity. Isolation and primary structure of equine growth hormone have been reported by Paladini and his coworkers (26, 27).

210

LI

TABLE

AND

CHUNG

I 50 %

PROTOCOLFORTHEISOLATIONOFEQUINEPROLACTIN

Fraction

Weight (9)

Procedure

30 %

A B C D E

Horse pituitary glands Acid-acetone powder NaCl fractionation Isoelectric precipitation Gel-filtration on Sephadex Chromatography on DEAE column

G-100

1000 40 11 2.7 0.51 0.13

proteins show the two negative bands characteristic of a-helical structure with negative maxima at 223 and 209 nm. The a-helical content of ePRL, as calculated according to Chen et al. (28), appears to be lower (50%) than that found in the ovine hormone (65%). Table II presents the amino acid composition of ePRL with a total of 199 residues. It has four half-cystines, two tryptophans, and four methionines. In order to ascertain the content of half-cystine and methionine, a sample of ePRL was oxidized with performic acid. The acid hydrolysate of the oxidized product was submitted to the automatic amino acid analyzer. Results showed that the oxidized sample had 4.3 cysteic acid and 4.2 me-

/-A 1 IO

0

I 20

30

Time (men)

FIG. 4. HPLC of ePRL on a reverse-phase 201 column (Alltech Assoc.; C1s, 300-A pore, nm); 30-50% linear gradient of propanol-2 ing 0.1% TFA; 30 min gradient; 1 ml/min; 1.0 AUFS.

VYDAC 4.6 X 250 contain210 nm;

thionine sulfone residues per molecule. These values are in agreement with that obtained using the native hormone (see Table II). Leucine was found to be the sole NH2terminal residue. By the dansyl-Edman procedure, the following NHz-terminal sequence was obtained: H-Leu-Pro-Ile-TrpLeu-. Digestion of the native hormone with carboxypeptidase Y with enzyme/ substrate ratio of l/10 in 0.1 M PyrAc, pH 5.5, for 8 h at 37°C did not liberate any amino acids indicating that the COOHterminal residue is not available for enzyme hydrolysis. However, when reducedcarbamidomethylated ePRL was digested by carboxypeptidase Y under the same

--

ePRL OPRL

Wavelength lnrnl

FIG. 3. Disc electrophoresis gel; pH 8.3; 3 mA per tube

of ePRL (50 rg); 7.0% (0.48 X 6 cm); 60 min.

FIG. 5. CD spectra HCI

buffer,

pH 8.2.

of ePRL

and oPRL

in 0.1

M Tris-

EQUINE

TABLE AMINO

ACID COMPOSITIONS Equine

Amino acid Asp Thr Ser Glu Pro % cys GIY Ala Val Met Ile Leu Tyr Phe His LYS -4% Tw Total

II OF EQUINE

PROLACTIN

prolactin Ovine prolactin (sequence)

Analysis

Integral value

22.5 3.7* 17.0* 29.9 7.8 3.9 7.2 11.1 9.9 3.6 10.8 26.8 6.1 5.9 6.7 8.2 14.1 2.0”

23 4 17 30 8 4 7 11 10 4 11 27 6 6 7 8 14 2

22 9 15 22 11 6 11 9 10 7 11 23 7 6 8 9 11 2

199

199

“Residues per molecule, based weight of 22,500 daltons. * Corrected. “Obtained from a methanesulfonic sate.

on

a molecular

acid

hydroly-

conditions, the amino acid analysis of the digest indicated that the hormone may have the following COOH-terminal sequence: -Leu-Val-Cys(Cam). The CNBr fragments of ePRL were fractionated on Sephadex G-50 in 0.1 M HOAc. As shown in Fig. 6, the reaction mixture (10 mg) was separated into three fractions with the following yields: I, 1.3 mg; II, 3.1 mg; and III, 2.5 mg. Fraction I was oxidized with performic acid and the oxidized product submitted to paper electrophoresis (Fig. 6). Fraction III was further fractionated by electrophoresis on paper (Fig. 6). The spots were cut out and the material was eluted with 0.1 M NHIOH solution. The eluates were dried in the dessicator for amino acid and NH,-terminal residue analyses. These analytical data are summarized in Table III. Summation of the content of each amino acid in the five

211

PROLACTIN

CNBr fragments is in agreement with the value for the amino acid composition of ePRL. It may be noted that the eluates from IIIA and IIIB (Fig. 6) has identical amino acid composition except that IIIA contains homoserine lactone (Hsl) whereas IIIB contains homoserine (Hse). This is also true for IIID (Hse) (Fig. 6). Table IV summarizes the assay data for ePRL as compared with that for the ovine hormone in the pigeon crop-sac test. It is evident that ePRL possesses the same potency of 42 IU/mg as oPRL (2). DISCUSSION

A purified ePRL has recently been obtained by Chen et al. (29) by serial extraction of horse pituitary glands followed by isoelectric focusing. These authors reported the hormone to have a potency of 35.6 IU/mg, isoelectric point of pH 5.8, and molecular weight of 25,000. In addition, they found that oPRL cross-reacts with the antiserum to ePRL. Using an entirely different method of isolation, we obtained a product with a potency of 42 IU/mg and a molecular weight of 22,500. Our procedure yielded 130 mg ePRL from 1 kg of glands whereas Chen et al. (29) reported a yield of 70 mg. No chemical data were presented by these investigators. From the NHz-terminal residue (Leu), it is evident that CNBr fragment IIIC is derived from the NHs-terminal segment

A.0

AS

C.DE

FIG. 6. Purification of CNBr fragments. Upper: gel filtration of the reaction mixture on Sephadex G-50 column (1.5 X 52 cm) in 0.1 M HOAc. Lower: paper electrophoresis (P.E.). Fraction I was oxidized with performic acid before electrophoresis.

212

LI

AND

CHUNG

TABLE AMINO

ACID

COMPOSITION

CNBr

OF PURIFIED

III FRAGMENTS

III Amino Cya Asp Thr Ser Glu Pro ‘h cys GUY Ala Val Met Ile Leu Tyr Phe His LYS Tw Arg Total residues NH,-terminal residues

acid

C

D

1.1 (1)

1.2 (1)

1.1 (1)

2.8 (3) 6.1 (6)

1.7 (2) 2.2 (2) 0.9 (1)

1.1 (1) 9.1 (9) 0.9 (1)

2.0 (2) (4) (1) (2) (2) (1)

2.0 (2) 0.7 (l)b 0.8 (1)

26

1.1 (1) 1.1 (1)

GUY

Da (1) 3.0 (3) 3.2 (3)

0.7 (1)

1.0 (1)

:, (1) 0.9 (1)

2.1 (2)

Leu

a Detected in the amino acid analyzer * Methanesulfonic acid hydrolysates. ‘Not detectable.

A

1.1 (1) 3.2 (3)

D” (1) 1.2 (1) 2.1 (2)

15

PROLACTIN

I

A

3.9 Da 1.5 1.9 0.9

FROM EQUINE

B

1.0 4.0 0.7 2.8 3.6 0.7

(1) (4) (1) (3) (4) (1)

1.1 1.9 1.6 Da 1.3 3.8 1.1 1.3 0.9 1.2

(1) (2) (2) (1) (1) (4) (1) (1) (1) (1)

2.1 (2)

26

31 Glu

2.9 13.6 2.8 8.5 13.7 3.1

(3) (14) (3) (9) (14) (3)

4.3 (4) 6.0 (6) 5.4 (5) 5.3 14.2 3.0 4.1 3.6 3.3

(5) (14) (3) (4) (4) (3)

7.3 (7)

101

N.D.’

Sum

ePRL

4 21 4 18 35 6

23 4 17 30 8 4 7 11 10 4 11 27 6 6 7 8 2 14

8 12 12 4 12 25 5 5 7 6 2 13

199

199

N.D.”

as Hse + Hsl.

of ePRL. Since fragment IB does not contain Hsl/Hse, it must be derived from the COOH-terminal protein of the hormone. Moreover, fragment IB is connected by -S-S bridge to the CNBr fragment IA. Exact arrangement for the CNBr fragments remains to be determined. In comparison with the prolactin from ovine and other mammalian species (2), ePRL has only four half-cystine residues. This is especially interesting as fish (Tilapia and salmon) prolactins also have four half-cystines (30-32). The three disulfide bonds of ovine PRL are formed by cystine-4-11 in the “amino-terminal loop,” by cystine-58-174, the “central disulfide bridge” and by cystine-191-199 which forms the “carboxyl-terminal loop” (2). It is apparent that the amino-terminal loop is missing in ePRL as shown by the NH,terminal sequence analysis and the amino

acid composition of CNBr fragment IIIC (Table III). The presence of the carboxylterminal loop in ePRL is evident as revealed by the action of carboxypeptidase Y on the reduced-carbamidomethylated hormone. The location of the central disulfide bridge in ePRL remains to be determined. The biological activity of ePRL is equipotent in comparison with the ovine hormone as assayed by the pigeon crop-sac test (Table III). Apparently, the loss of the amino-terminal loop does not alter the lactogenic potency. This is consistent with observations that reduction of cystine-411 in ovine PRL retains full biological activity (33) and that salmon PRL5 which 5The terminal Met-.

salmon PRL (32) has the following sequence: NHz-Ile-Gly-Leu-Ser-Asp-Leu-

NHa-

EQUINE

TABLE BIOLOGICAL ACTIVITY IN THE PIGEON Prolactin

Total

PROLACTIN

IV

OF EQUINE PROLACTIN CROP-SAC ASSAY dose (pg)

Response”

Ovine

2

22.4 t 1.6

Equineb

6 2 6

35.8 f 1.2 23.3 + 2.5 26.1 f 4.1

” Dry mucosal weight in mg; mean + standard error from three birds in each ease. * Relative potency to oPRL (42 IU/mg; see Ref. (a)), 105% with 95% confidence limit of 62-179, and X = 0.25.

does not have the NHz-terminal loop has the same pigeon crop-sac stimulating activity as the ovine hormone (32). It appears that the amino-terminal loop is not essential for hormonal function of the prolactin molecule.

11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

ACKNOWLEDGMENTS We thank Dr. Thomas A. Bewley for the CD data and Jonathan Roeder and J. D. Nelson for technical assistance. This work was supported in part by grants from the National Institutes of Health (AM6097) and the Hormone Research Foundation.

24.

25. 26.

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