Immunochemical investigations of porcine lactogenic hormone

Immunochemical investigations of porcine lactogenic hormone

ARCHIVES OF BIOCHEMISTRY AND lmmunochemical BIOPHYSICS Investigations W. CRAIG Hormone Reseaxh 161, 313-318 Laboratory, of Porcine CLARKE?...

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Investigations W. CRAIG



161, 313-318


of Porcine

CLARKE? University Received





of Califortria, October


Sal1 Fratrcisco,



3, 1973

Guinea pig ant,isera to porcine prolactin were characterized by immunodiffusion, quantitative precipitin, and microcomplement fixation techniques. In gel double diffusion tests, the ant,isera cross-reacted with ovine and bovine prolactins, but not with rat prolactin, porcine growth hormone, or human growth hormone. Microcomplement fixation experiments indicated a considerable degree of immunological similarity among porcine, ovine, and human prolactins. Modification of porcine prolactin either bv reduction or oxidation decreased its immunoreactivity and abolished its biological acivity in the pigeon crop-sac assay.

The recently elucidated (1) primary structure of porcine prolactin showed a high dcgree of homology with oPRL.~ Porcine prolactin has been shown to cross-react wit’h antisera to oPRL (2) and hPRL (3), but not with antisera to mPRL (4). The present investigation was undertaken to characterize immunochemically guinea pig antisera to pPRL, and to obtain more informat’ion concerning the structural similarity of pPRL to other prolactins and hormonal proteins having lactogenic activity. In addition, the ability of the antisera to rccognizc oxidized and reduced pPRL n-as investigated in order to evaluate the function of the disulfidc bridges in conferring immunochemical specificit,y upon the prolactin molecule. 1 Paper XXXV in the Studies on Pituitary Lactogenic Hormone series. This work was supported in part by U.S. Public Health Service Grant AM6097. 2 Recipient of a postdoctoral fellowship from the Medical Research Council of Canada. 3 Abbreviations used are: pPRL, porcine prolactin; oPRL, ovine prolactin; bPI(L, bovine prolactin; hPRL, human prolactin; rPI:L, rat prolactin; mPRL, mouse prolactin; p(;H, porcine growth hormone; hGH, human growth hormone; hCS, human chorionic somatomammotropin; RCAM-pPRL, reduced-carbamidomethylatedportine prolactin. 313 Copyright All rights

@ 1974 by Academic Press, of reproduciirm in any form

Inc. reserved.



Materials. Prolactin was isolated from pig pituitary glands by procedures essentially the same as those previously described for oPRL (5). Human growth hormone was prepared by the method of Li et al. (6). Human prolactin was kindly provided by Dr. U. J. Lewis (7). Rat prolactin was a gift of the National Institute of Arthritis, Metabolism, and Digest,ive Diseases. Procedures for preparation of the performic acid oxidized and reduced-carbamidomethylated prolactin have been reported elsewhere (8, 9). Amino acid analyses were performed by the method of Spackman et al. (10) in an automatic amino acid analyzer (Beckman Instnlment Co., Palo Alto, CA). Purified pPRL was dissolved in 0.01 M phosphate buffer (pH 7.5) and emulsified with an equal volume of complete Freund’s adjuvant (Difco). Male guinea pigs were given 5 injections of 0.2 mg each over a period of 6 weeks, for a total dose of 1 mg per animal. One week after the final injection, blood was withdrawn by cardiac punct,ure and allowed to clot. Serum was prepared as outlined by Kabat and Mayer (11). Biological neutralizatio,l. Antiserum was tested for its ability to inhibit the response to a 4 pg dose of pPRL in the local pigeon crop-sac assay (12). The antisertlm was allowed to react with the hormone for 1 hr at 37”C, left to stand 72 hr at 4”C, and centrifuged to separate the precipitate. The supernatant was then decanted, and the precipitat,e was resuspended in saline for injection. Immunochemid methods. The gel double dif-



fusion technique (13) was performed using 1% agar in 0.01 M phosphate buffer (pH 7.5). The procedure for quantitative precipitin tests was essentially that of Kabat and Mayer (11). The reaction volume was 1 ml, with incubations at 37°C for 1 hr and at 4°C for 72 hr. Total protein precipitated was determined by the method of Lowry el al. (14). Microcomplement fixation experiments were performed according to Wasserman and Levine (15). The diluting buffer (0.14 M NaCl buffered at pH 7.5 with sodium diethylbarbital) contained 1.5 X 1V M calcium chloride, 5 X 1e4 M magnesium chloride, and 0.1% bovine serum albumin. Fixation of complement was conducted in a volume of 1 ml for 14 hr at 4°C. The volume was increased to 7 ml by the addition of activated sheep erythrocytes, and the tubes were incubated for 1 hr at 37°C. After cooling to 0°C and centrifugation to remove the unlysed cells, the absorbance of the supernatant solution was measured at 413 nm.



tion of complement (Figs. 2B and 3A). HOW ever, if the concentration of antiserum GP4 was increased 6.5 times, it reacted similarly with either hPRL or oPRL to fix X0-90% complement (lcig. 3B). Antiserum Cl’8 at a


Double d$u.sion. Antisera to pPRL produced a single, sharp precipitin line against l-2 pg of the hormone or against crude extracts of pig pituitaries. Reduced-carbamidomethylated porcine prolactin also gave a single precipitin line which showed a reaction of qualitative identity with the native hormone, although 10 times as much antigen was needed to give a visible line. On the other hand, oPRL, bPRL, and performic acid oxidized pPRL showed partial identity with native pPRL, while pGH, hGH, rPRL, and crude extracts of rostra1 pars distalis from the teleost Tilapia mossambica failed to yield precipitin lines. Results of some of the immunodiffusion studies are shown in Fig. 1. Quantitative precipitin. The precipitin curve obtained with antiserum GP8 to pPRL is given in Fig. 2A. The homologous antigen precipitated 165 pg of protein at 10 pg of antigen, whereas oPRL precipitated only 62 Hg protein at the same antigen concentration. Microcomplement jixation. This technique distinguished oPRL from pPRL more readily than did the precipitin assay. Antisera GP4 and GP8 reacted with pPRL to fix 8O-100% complement at dilutions of 1: 2500 and 1:5000, respectively, but when reacted with oPRL at the same antiserum concentration there was virtually no fixa-

FIG. 1. Immunodiffusion plate showing precipitin reactions of various hormones with guinea pig antiserum to porcine prolactin. (A) Center well, 50 ~1 antiserum GP4; (1) 50 /rg pPRL; (2) 50 rg oPRL; (3) 50 /*g bPRL; (4) 50 pg pGH; (6) 50 pg hGH; (6) 500 rg porcine pituitary homogenate. (B) Center well, 20 ~1 antiserum GP8; (1) 10 pg pPRL; (2) 70pg RCAM-pPRL; (9) 70 pg performic acid oxidized pPRL.




FIG. 2. (A) Quantitative (e-0) curves

or oPRL (O-O); obtained with pPRL









precipitin reaction of ant,iserum GPB to pPRL with pPRL 50 ~1 of antiserum per tube. (B) Microcomplement fixation (o-0) or oPRL (O--O) and antiserum GP 8 diluted








1 ug

FIG. 3. (A) Microcomplement fixation curves obtained with pPRL ([email protected]) (0-O) and antiserum GP4 diluted l/2500. (B) Microcomplement fixation tained with the same antiserum diluted l/400 and oPRL (O---O) or hPRL

dilution of 1: 1200 also reacted with hPRL to fix 90 % complement. Moreover, at a concentration 5 times that required for the homologous reaction, antiserum to oPRL reacted with both pPRL and hPRL to fix complement (Fig. 4). Thus, it appears that pPRL, oPRL, and hPRL have a number of antigenic determinants in common. On the other hand, no complement fixation was observed when hGH was reacted with antiserum GP4 at a dilution of 1:250. Reduction and alkylation of the cystine


or oPRL curves ob(A-A).

residues in pPRL caused a shift in the zone of maximal complement fixation to regions of higher antigen concentrations, although no increase in antiserum concentration was required (Fig. 5A). However, performic acid oxidation produced a profound aheration in the immunochemical properties of pPRL so that comparable complement fixation could not be demonstrated until the antiserum concentration was increased 8 times (Fig. 5B). dmino acid analyses. The results are shown in Table I. Both RCAM-pPRL and



performic acid oxidized pPRL lacked halfcystine (i.e., less than 0.1 residue), indicating essentially stoichiometric conversions to carboxymethylcystine and cysteic acid, respectively. Moreover, performic acid oxidized all 4 methionine residues to methionine sulfone. Biological activity. The data presented in Table II show that 1 pg pPRL caused a proliferation of the pigeon crop-sac mucosa, whereas 40 pg of RCAM-pPRL and performic acid oxidized pPRL had no statis-


s .05









FIG. 4. (A) Microcomplement fixation curves obtained with oPRL (O---O) or pPRL (O--O) and guinea pig antiserum to oPRL diluted l/7500. (B) Microcomplement fixation curves obtained with the same antiserum diluted l/1500 and pPRL (A--A). (0-O) or hPRL



tically significant effect. There was no significant activity in either the precipitate or supernatant fraction after the reaction of 4 pg pPRL with antiserum GP8. TABLE I AMINO ACID COMPOSITION OF PORCINE PROLACTIN AND DERIVATIVES~ Amino acid

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half-cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Carboxymethylcysteine Cysteic acid Methionine sulfone





8.3 12.8 22.1 5.1 13.6 24.2 7.2 8.5 10.2 4.9 10.1 4.0 13.0 24.5 7.0 6.2 0

8.4 8.0 12.1 22.3 5.3 15.7 24.9 7.5 8.5 10.8 0 10.2 4.3 12.9 24.8 7.3 6.3 6.5

8.4 7.8 12.2 22.0 5.1 15.3 24.2 7.8 8.4 10.5 0 10.8 0 12.9 26.3 6.8 6.7 0

0 0

0 0

5.4 4.3

4 RCAM-Prolactin, reduced-alkylated porcine prolactin; PO-Prolactin, performic acid oxidized porcine prolactin.





FIG. 5. (A) Microcomplement fixation curves obtained with pPRL (O---e) or RCAMpPRL (O--U) and antiserum GP8 diluted l/8000. (B) Microcomplement fixation curve obtained with antiserum GP8 diluted l/1000 and performic acid oxidized pPRL.






Saline cont,rol PRL

Total dose iA -~-__ 0 1 4

Dry mucosal wt” (md


9.0 It 0.4 18.8 zt 0.6e 23.0 f l.le


10 40

12.8 f 13.3 f

2.2 2.0


10 40

8.3 f 13.2 f

1.3 1.6


9.9 & 1.7


9.8 f


6 Mean + standard error; four test animals in each group. b Reduced-alkylated porcine prolactin. L Performic acid oxidized porcine prolactin. d Fractions obtained after precipitation of 4 pg pPRL with 0.03 ml antiserum. 6 Significantly different from saline cont.rol (p < 0.01) by Dunnett’s one-sided comparison test. DISCUSSION

The results of the biological ncutralization, gel double diffusion, quantitative prccipitin, and microcomplement fixation experiments demonstrated the specificity of the antisera for pPRL. Their distinct, but reduced, affinity for oPR,L is not surprising in view of the difference in primary structure of the two hormones (1, 5) and the previous demonstration of a cross-reaction between pPRL and antisera to oPRL in a radioimmunoassay (2). The antisera failed to cross-react with rPRL in gel double diffusion test’s in accordance with earlier im munodiffusion studies using ant’isera to oPRL (16, 17). Also, antisera to rPRL did not cross-react with oPRL or pig pituitary extracts (18). Our demonstration of a crossreaction with hPRL is in agreement with the findings of Sinha et al. (3). They reported tllat, 1 pg pPRL giws a reaction of partial identitg with antisera to hPRL in immunodiffusion tests, but dots not cross-react in a radioimmunoassay at antiserum conrentrations used for t’he homologous antigen dat’a (hPRL). These immunochemical



pointrd to a homology between pPRL, oPRL, and hPRL, and corroborated prcliminary reports (19, 20) that hPRL is rather similar to oPRL in terms of its amino acid composition. On the other hand, hGH failed to fix complcmcnt at’ even higher antiserum concentrations, indicating that it is immunochcmically distinct from hPRL (3, 21). The antisera failed to product a prccipitin line against pituitary extracts from the teleost fish Tilapia mossambica, which is knolvn to be rich in prolactin (22). There are accounts of a cross-reaction between antisera to oPRL and extracts of pituitaries from several telcost species (23-X). The microcomplement fixation technique was considerably more sensitive to differences in protein structure than either the gel double diffusion or quantitative prccipitin techniques. It) detected not only differences in the amino acid composition of proteins, but also the conformational changes induced by cleavage of the disulfide bridges (27-31). Reduction and carbamidomethylation of the cgstine residues in pPRL decreased the amount of complement fixed at equivalence, and shifted the curve t#othe region of higher antigen concentration. Reduced-carbamidomethylated porcine prolactin lacked pigeon crop-sac act’ivity, in contrast to RCAMhGH which retained both its crop sac and growth promoting activities (32, 33). These data indicat’e the disulfidc bridges are more necessary for maintaining the conformation of pPRL than that of hGH. Nevertheless, physicochcmical studies (33) have shown RCA&l-hGH to have a slightly alt’ercd secondary a,nd tertiary structure which is rcfleeted in a 30 % decrease in its reactivity in radioimmunoassay (34). Performic acid oxidation also destroyed the crop-sac activity of pPRL, but resulted in a more drama& decrease in its ability to fix complement than did reduction and alkylation. Trenklc et al. (27) previously demonstrated an impaired ability of hGH to fix complement after pcrformic acid oxidation, and scvcral recent investigations have obtained similar decreases in immunoreactivity following reductive or oxidativc cleavage of the disulfidc linkages of hGH and hCS (31,353s).




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