Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 3540-3544, May 1991 Medical Sciences Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami (proopiomelanocortin precursor/neurophysin precursor/prolactin release-inhibiting factor) ANDREW V. SCHALLY*t, JANOS G. GUOTH*t, TOMMIE W. REDDING*, KATE GROOT*, HENRY RODRIGUEZf, EVA SZONYIt, JOHN STULTS*, AND KAROLY NIKOLICSt *Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, 1601 Perdido Street, New Orleans, LA 70146; tDepartment of Experimental Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112; and tDepartments of Developmental Biology and Protein Chemistry, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080 Contributed by Andrew V. Schally, December 28, 1990

ABSTRACT Two peptides with in vitro prolactin release- Here we report the isolation of two peptides from porcine inhibiting activity were purified from stalk median eminence hypothalami, different from gonadotropin releasing hor- (SME) fragments of 20,000 pig hypothalami. Monolayer cul- mone-associated peptide (GAP) and somatostatin-14 (SS-14), tures ofrat anterior pituitary cells were incubated with aliquots that exhibited a dose-dependent prolactin release-inhibiting of chromatographic fractions and the inhibition of release of activity in vitro. These substances or their congeners might prolactin in vitro was measured by RIA in order to monitor the play a physiological role in the regulation ofprolactin release. purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in MATERIALS AND METHODS prolactin release-inhibiting factor (PIF) activity. This material Isolation of the two peptides with PIF activity from 20,000 was further purified by semipreparative reversed-phase (RP) lyophilized stalk median eminence (SME) fragments of pig HPLC. This process resulted in the separation of two distinct hypothalami (Oscar Mayer, Madison, WI) was accomplished fractions that showed high PIF activity. These were further essentially by sequential purification in six steps. After each purified by semipreparative and analytical RP-HPLC to ap- purification step, the fractions were pooled and their in vitro parent homogeneity as judged by the UV absorbance profiles. PIF activity and levels of immunoreactive GAP and SS-14 Neither of the two peptides showed cross-reactivity with go- were determined. nadotropin releasing hormone-associated peptide or with so- Extraction. Extraction was carried out as described in matostatin-14 antibodies. Protein sequence analysis revealed detail by Schally et al. (3). Briefly, lyophilized fragments of that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser- 20,000 pig hypothalami, weighing 531 g, were first pulverized, Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-Ala- defatted by acetone and petroleum ether, extracted with 2 M Cys-Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of acetic acid at 80C, and centrifuged (3). Phenylmethylsulfonyl the N-terminal region of the proopiomelanocortin (POMC) fluoride and pepstatin A (10 pug/ml each) were added to the precursor (corresponding to amino acids 1-26 of the 16-kDa clear supernatant (14). The mixture was heated to boiling, fragment). The sequence of the other PIF was Ala-Ser-Asp- immediately cooled on ice to 40C, and centrifuged. The clear Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala- supernatant was lyophilized, resulting in 114.5 g of dry Leu-Leu-Leu-Arg-Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu- extract from porcine hypothalami. Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr, representing res- Preparative Size-Exdusion HPLC. From the lyophilized idues 109-147 of the vasopressin-neurophysin precursor. hypothalamic extract, 98 g (16,200 SME) was dissolved in Synthetic peptides corresponding to the N-terminal region of 50% acetic acid (71.5 ml), diluted with distilled water to 4290 POMC had significant PIF activity in vitro. ml (final pH 2), and centrifuged (Sorvall RC S B; 26,890 x g; 30 min). The clear supernatant was then subjected to high- The existence ofprolactin release-inhibiting factor(s) (PIF) in performance aqueous size-exclusion chromatography on a rat hypothalamic extracts was first demonstrated by Pasteels TSK G-2000SW (21.4 x 600 mm) column (Toyo-Soda, Phe- (1) and Talwalker et al. (2). Several substances were later nomenex, Rancho Palos Verdes, CA), after equilibration of identified in mammalian hypothalamic tissues that inhibited the stationary phase with 10 bed vol of 0.1 M NaCl/0.05 M the release of prolactin (3-7). In humans, the administration Tris'HC1, pH 4.4. The flow rate of the mobile phase was 6 of dopamine receptor antagonists, such as chlorpromazine ml/min. In total, 88 separate chromatographic runs were and other neuroleptics, markedly increases prolactin level, performed under identical conditions. In each run, 11 frac- whereas dopamine agonists, such as the ergot derivatives, tions were collected (Fig. la). significantly reduce plasma prolactin concentration (8). Preparative Reversed-Phae (RP) HPLC. Fraction JGG- Based on these clinical and experimental data, dopamine was 2-53 no. 3/1 in aliquots of 90 ml from the TSK G-2000SW considered to be the only physiological PIF. column, exhibiting significant in vitro PIF and immunoreac- On the other hand, peptidic substances have been partially tive GAP activities, was subjected to RP chromatography on purified from brain extracts, which also had significant PIF a Dynamax C18 preparative column (Rainin Woburn, MA) activity (9-12). Our preliminary results also suggested that (250 x 41.4 mm; 12-pum particle size; 300-X pore size). A dopamine is not the only hypothalamic substance with PIF linear gradient was used for the elution of the substances activity (13). However, the isolation of a highly potent adsorbed to the matrix of the column. Component B of the peptide PIF from hypothalamic extracts has not yet been mobile phase was increased from 0% to 10%o in 70 min accomplished. Abbreviations: PIF, prolactin release-inhibiting factor; POMC, proo- The publication costs of this article were defrayed in part by page charge piomelanocortin; GAP, gonadotropin releasing hormone-associated payment. This article must therefore be hereby marked "advertisement" peptide; RP, reversed-phase; SS-14, somatostatin-14; SME, stalk in accordance with 18 U.S.C. §1734 solely to indicate this fact. median eminence. 3540 Downloaded by guest on September 29, 2021 Medical Sciences: Schally et A Proc. Natl. Acad. Sci. USA 88 (1991) 3541 increased from 35% to 70% in 140 min, while using a flow rate of 8 ml/min (Fig. 1c). Twenty-four fractions (JGG-2-149) IN 0.5. were collected and Iyophilized. A 16-mg aliquot of the material with the highest in vitro PIF activity (JGG-2-149 no. -Ce 14) was further purified on a Vydac C18 (Rainin) 250 X 10.0 size and 300-A size 0 10 mm, 5-pum particle pore semipreparative 1 20 30 RP column, using the same mobile phase as in preparative b RP-HPLC. A flow rate of 2 ml/min and a shallow linear was 0 gradient used. Component B of the mobile phase was " 50- increased from 40% to 62% in 72 min (Fig. ld). Twenty-five fractions were collected and Iyophilized (JGG-2-189). A 6-mg fraction with the highest PIF activity (JGG-2-189 0 40 80 nos. 17 and 18) was rechromatographed on the same Vydac Time, min C18 RP column as in step III using the trifluoroacetic acid/ Scheme A F 12 1 13 Scheme B acetonitrile/water, mobile phase, with a shallow linear gra- dient, increasing component B of the mobile phase from 35% g to 70% in 140 min (Fig. le). The separation resulted in 27 fractions (JGG-7-29). gj 0.5- The most potent PIF fraction (JGG-7-29 no. 12), weighing 71 pAg, was subjected to purity tests on an Aquapore RP-300 0.0 (Brownlee, Phenomenex), 250 x 1.0 mm, 7-pum, 300-A mi- 0 40 80 0 40 80 crobore a 1.0d 1.0 column, using linear gradient (solvent A, 0.1% o iiri trifluoroacetic acid/water; solvent B, 0.1% trifluoroacetic N-0 acid/70% acetonitrile/30%o water) (Fig. if), with,a flow rate of 0.08 ml/min. Component B of the mobile phase was increased from 40% to 52% in 40 min. The fraction was 0.0 subjected to protein sequencing. 0 40 80 20 40 Purification Scheme B. The second fraction from prepara- 1.OTr- tive RP-HPLC with high in vitro PIF activity (JGG-2-127 no.

0 13) was dissolved in 50%o solvent A/50% solvent B, filtered " 0.5 through a hydrophilic, chemically resistant, 25-mm nylon -ce Acrodisc filter unit (0.2-pmm pore size) (Gelman) and sub- jected to repeated separation on the same preparative column 0.0 - 0I 10 20 and in the same mobile phase as described in the previous RP Time, min separation step. However, the linear gradient was designed to be shallow. Component B of the mobile phase was increased from 40% to 62% in 65 min. Sixty fractions were collected and lyophilized (JGG-2-219) (Fig. ig). Fraction JGG-2-219 no. 30 with the highest in vitro PIF 10 activity was further purified on a W-Porex 5C18 (Phenom- Time, min enex) (250 x 4.6 mm; 5-,um particle size; 300-A pore size) analytical RP column, using the same mobile phase as in FIG. 1. Chromatographic purification of the two peptides with preparative RP separation. A flow rate of 1.2 ml/min and a prolactin release-inhibitory activity. Extracts of 20,000 porcine hy- shallow, linear gradient was used. Component B of the pothalami were chromatographed in successive steps by size- mobile phase was increased from 42% to 64% in 40 min. exclusion HPLC (a) and preparative RP-HPLC (b). The shaded or Fifteen fractions were collected and Iyophilized darkened areas represent fractions with the highest in vitro prolactin (JGG-7-169) release-inhibitory activity that were collected and processed in (Fig. lh). subsequent steps. Fraction 12 of b (JGG-2-127) was further chro- The most potent fractions in the PIF bioassay, JGG-7-169 matographed by preparative and analytical RP-HPLC (c-f). Fraction no. 10, weighing 200 pLg, was subjected to a purity test on 13 of b was further chromatographed by RP-HPLC (g-i). The shaded Aquapore RP-300 under the same conditions as in scheme A areas of f (JGG-7-29-12) and darkened areas of i (JGG-7-169-10), (Fig. ii). respectively, were analyzed by protein sequencing and mass spec- All the separation procedures were performed at room trometry. temperature, but the fractions were collected and kept at 40C. The UV absorbance of the fractions eluted from the columns (solvent A, 0.1% aqueous trifluoroacetic acid; solvent B, was measured at 220 and 280 nm. For the preparative 0.1% aqueous trifluoroacetic acid in 70o acetonitrile), while purification process, a Beckman HPLC system (Beckman, using a flow rate of 62 ml/min (Fig. lb). Sixteen fractions Berkeley, CA) with a 450 data system controller, two 114 M (JGG-2-127) were collected and pooled from each of the 16 solvent delivery modules, a 340 organizer, a 165 variable separate runs. wavelength detector, a Kipp and Zonen BD41 recorder, and Purification Scheme A. One of the two fractions with the a Gilson model 201 fraction collector or a MacRabbit HPLC highest in vitro PIF activity (JGG-2-127 no. 12), weighing 100 system (Rainin), with a Knauer UV photometer, a Gilson mg, was dissolved in 50%o solvent A/50% solvent B and model 202 fraction collector, a Kipp and Zonen BD41 re- filtered through a hydrophilic, chemically resistant, 25-mm corder were used. For the analytical steps, a HP-1090 liquid nylon Acrodisc filter unit (0.2-pum pore size) (Gelman). It was chromatograph HPLC system (Hewlett-Packard) was used. then subjected to RP chromatography on a Dynamax C18 The solvents were HPLC grade, and the reagents were (Rainin) (250 x 21.4 mm; 12-,um pore size; 300-A particle HPLC or analytical grade in purity, obtained from Burdick size) preparative column. The same mobile phase was used and Jackson, Sigma, and from Fluka. Distilled water was also as in the previous preparative RP separation step, with a purified through a Milli-Q water purification system (Milli- different gradient. Component B of the mobile phase was pore). Downloaded by guest on September 29, 2021 3542 Medical Sciences: Schally et al. Proc. Natl. Acad. Sci. USA 88 (1991)

Prolactin and SS-14 RIA. RIA for prolactin was carried out Table 1. HPLC purification scheme for a porcine hypothalamic with materials supplied by the National Hormone and Pitu- peptide (JGG-7-29 no. 12) with PIF activity in pituitary itary Program (National Institute of Diabetes and Digestive cell culture and Kidney Diseases). The prolactin concentrations in the Prolactin samples were measured in duplicate by a double antibody RIA method, using as standard rat prolactin, the RP3 refer- Total SS-14, GAP, release* ence preparation. The statistical significance was assessed by Sample wt pg/SME ng/SME 4 hr 24 hr Duncan's new multiple range test. For the RIA of SS-14 (15) 20,000 pig JH 204 antibody was used. hypothalami 531 g GAP RIA. Substances with GAP immunoreactivity were AVS-10-122 114.5 g determined by a RIA method, using the KN-16 antibody in a 2 M acetic acid 1: 40,000 final dilution (16). The iodination ofthe antigen was extraction performed by the chloramine-T method. The labeled hor- JGG-2-53 no. 3/1 2.81 37 74 mone was repurified by gel filtration on a Sephadex G-50 JGG-2-127 no. 12 100 mg 12,278 1.86 41 51 column. The specific activity of the labeled hormone was JGG-2-149 no. 14 16 mg 1,487 1.24 37 49 1375 ,uCi/,ug (1 Ci = 37 GBq). The standard curve was set up JGG-2-189 nos. 17 + 18 1 mg 5,041 18.7 21 33 in the range between 0.10 pg/1lt and 1 ng/,pi. The binding of JGG-7-29 no. 12 131 ,ug 0 0 58 39 the labeled hormone to the was antibody 28%.' The interassay The duration ofthe incubation ofthe cells with the tested materials and intraassay variations were less than 12% and 10%, was 4 and 24 hr in each experiment. respectively. *Decrease in prolactin release, % control. PIF in Vitro Bioassay. The monolayer culture assay will be reported in detail elsewhere. Briefly, anterior pituitaries from The TSK preparative size-exclusion HPLC concentrated donor female rats weighing 200-250 g were removed asepti- PIF activity into 11 fractions. JGG-2-53 no. 3/1 was found to cally and dispersed with 0.3% collagenase in Dispase (50 be one ofthe most potent compounds showing 74 units ofPIF units/ml) and DNase (10 pug/ml). Cells were washed twice activity per SME and 576 units ofPIF specific activity per mg, and 0.3 x 106 cells were plated per well in 24-well culture in vitro. This heterogenous fraction was concentrated and dishes in Dulbecco's modified Eagle's medium (DMEM) plus separated on a preparative C18 reversed-phase column. The 10% fetal calf serum. Cells were incubated at 370C in 5% process resulted in three fractions with significant PIF activ- C02/95% air for 5 days before they were used in the assay ity. The greatest biological activity was found in the 100-mg system. On the day of the assay, cultures were washed twice fraction designated JGG-2-127 no. 12, with 51 units of PIF with DMEM without serum. Samples dissolved in this same activity per SME or 2040 units ofPIF activity per mg in vitro. medium were added as 1-ml aliquots to each of four wells. The chromatography performed on a C18 column using a Cultures were incubated an additional 24 hr and aliquots of shallow gradient (0.25% solvent B increase per min) produced the medium were taken after 4 and 24 hr for determination of fraction JGG-2-149 no. 14, weighing 16 mg. This fraction prolactin levels by RIA. PIF activity was arbitrarily defined showed 49 units of PIF activity per SME or 6125 units of PIF as the prolactin release-inhibiting activity ofthe compound as activity per mg and was still heterogenous (Table 1). compared to the effect of the control material in the same After purification on another C18 column, two fractions bioassay. The effect was expressed as the percentage of (JGG-2-189 nos. 17 and 18) were obtained (Fig. ld), activity of the control material in units per SME or units/mg. weighing Since catecholamines and SS-14 contribute to PIF activity in 1 mg and exhibiting 33 units ofPIF activity per SME or 16,500 extracts in this the in units units of PIF activity per mg. Fraction JGG-2-189 no. 17 hypothalamic assay, PIF activity a per SME was at times decreased after a purification step due exhibited dose-dependent PIF effect in vitro (Table 1) but to the elimination of these substances. showed significant cross-immunoreactivity with SS-14 and Protein Sequencing. Purified fractions of the final chro- GAP in the RIA (Table 1). matographic runs were analyzed by N-terminal protein se- The final analytical chromatography using acetonitrile/ quencing. Automated Edman degradation was performed trifluoroacetic acid/water, mobile phase, with a shallow with an Applied Biosystems model 470A gas-phase se- gradient (0.25% solvent B increase per min) resulted in 71 ,ug quencer equipped with a 120A phenylthiohydantoin amino of fraction JGG-7-29 no. 12. An estimated 59.5 ,ug of this acid analyzer. Phenylthiohydantoin-derivatized amino acids material was used up for the RIAs and bioassays to guide the were identified by RP-HPLC and integrated with a Nelson purification. Considering all these data, the yield of the analytical model 3000 data system. Sequence interpretation HPLC purification ofJGG-7-29 no. 12 was 130.5 ,ug or 91.2%. was performed on a Vax 11/785 computer (Digital Equip- The isolated material exhibited a strong UV absorption at 220 ment) as described (17). nm and a lesser one at 280 nm and showed 93% homogeneity Electrospray Ionization Mass Spectrometry. Electrospray as assessed by UV absorbance (Fig. 1f) and had a PIF ionization mass spectra were obtained with a Sciex API III activity estimated at 39 units per SME or 55,715 units/mg. triple quadrupole mass spectrometer (Thornhill, Ontario, JGG-7-29 no. 12 was found to be the most potent PIF fraction Canada), using a pneumatically assisted Ionspray nebulizer. in the 24-hr incubation test ofthe pituitary cells. This material The peptide was dissolved in 10% formic acid with water/ acetonitrile (1:1), and it was pumped into the nebulizer (5000 Table 2. HPLC purification scheme B of a porcine hypothalamic peptide (JGG-7-169 no. with PIF V) at 5 1L/min. The orifice was maintained at 120 V. The mass 10) activity axis was scanned from 600 to 1600 units (u) in 15 sec using Prolactin 0.2-u steps. Total SS-14, GAP, release* Sample wt pg/SME ng/SME 4 hr 24 hr RESULTS JGG-2-127 no. 13 1094 0.65 35 30 The 2 M acetic acid extraction of 20,000 pig hypothalami JGG-2-219 no. 30 810 5.30 4 27 resulted in a yield of 114.5 g of dry powder. For isolation of JGG-7-169 no. 10 240 ,ug 0 0 13 30 fractions with PIF activity, 98 g ofthis material was subjected , Not available. Incubation periods of4 and 24 hr in pituitary cell to purification by various HPLC methods (Table 1 and culture were used for the bioassay. Table 2). *Decrease in prolactin release, % control. Downloaded by guest on September 29, 2021 Medical Sciences: Schally et A Proc. Natl. Acad. Sci. USA 88 (1991) 3543 exhibited no cross-reactivity in the RIAs for SS-14 or GAP. Table 3. PIF activity of purified hypothalamic PIF and rat, This chromatographic procedure resulted in a 96.7-fold in- bovine, and human POMC fragments in the pituitary cell crease in PIF activity when assayed in the pituitary cell culture bioassay culture system (Table 1). This fraction was subjected to Prolactin structural analysis. Fraction JGG-2-127 no. 13 with high PIF activity was Dose,* ng/ml releaset purified separately according to scheme B (Fig. 1 g-i). The Sample ,ug At 4 hr At 24 hr 4 hr 24 hr chromatography performed on a Dynamax C18 column but Control - 1730 ± 70 11,413 ± 400 - using a shallow gradient (0.25% solvent B increase per min) JGG-2-189 resulted in JGG-2-219 no. 30 as the most potent fraction. The no. 17 0.1 1330 ± 60 7,800 ± 600 23f 32t final semipreparative chromatography, using acetonitrile/ JGG-2-189 trifluoroacetic acid/water, mobile phase, but shallow gradi- no. 17 0.5 1187 ± 170 4,075 ± 600 31§ 64§ ent (0.25% solvent B increase per min) on a W-Porex C18 Control - 1040 ± 22 5,850 ± 150 - column, results in 240 Ag of JGG-7-169 no. 10. An estimated Rat 0.01 1000 ± 26 4,630 ± 88 4 21§ 40 pug of this substance was used for the RIAs and bioassays POMC 0.1 920 ± 19 3,730 ± 312 11t 36§ to guide the purification procedures. Thus, the yield of the 1-49 1.0 790 ± 27 3,230 ± 78 24§ 45§ HPLC purification of JGG-7-29 no. 12 was 91.2%. The Bovine 0.01 930 ± 17 4,580 ± 287 11t 22§ isolated substance exhibited strong UV absorption at 220 nm, POMC 0.1 960 ± 14 4,950 ± 215 8 15t no UV absorption at 254 nm, and low absorption at 280 nm, 1-49 1.0 800 ± 38 4,240 ± 317 23§ 28§ and a PIF activity of 17,391 units/mg. JGG-7-169 no. 10 was Human 0.01 740 ± 64 4,200 ± 235 29§ 28§ found to be a potent fraction showing PIF activity after a 4- POMC 0.10 920 ± 96 4,280 ± 127 11t 27§ and a 24-hr incubation period in the pituitary cell culture 1-49 1.0 800 ± 10 4,690 ± 120 23§ 20§ bioassay. The material showed no cross-reactivity to SS-14 *Dry weight. or GAP by RIA. tDecrease in prolactin secretion release, % control. This chromatographic procedure resulted in a 30.1-fold tSignificantly different from control; P < 0.05. increase in PIF specific activity in the pituitary cell culture §Significantly different from control; P < 0.01. system (Table 2). Fraction JGG-7-169 no. 10 was subjected to protein sequence analysis by Edman degradation. DISCUSSION Protein Sequencing and Mass Spectrometric Analysis. An aliquot of fraction JGG-7-29 no. 12 was subjected to protein The hypothalamic control of pituitary prolactin release is sequencing for 26 cycles. The sample was found to contain as mediated by stimulatory and inhibitory agents (8). Several the highest signal Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln- compounds are known to inhibit prolactin release in pharma- Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-Ala-Cys-Ile-Arg- cological doses, such as dopamine, norepinephrine, y-ami- Ala-Cys-Lys-Pro. The sequence corresponds to residues nobutyric acid, and acetylcholine (13). Purification of various 27-52 of the N-terminal region of the porcine proopiomel- fractions with PIF activity from hypothalamic extracts in our anocortin (POMG) peptide and residues 1-26 of the 16-kDa laboratory led to the isolation ofdopamine and norepinephrine fragment. Underlying this sequence, another signal was (6), as well as y-aminobutyric acid (7). Dopamine is present in detected in every cycle, which corresponded to the N-ter- a high concentration in median eminence (18) and in hypophy- minal region of the a chain of porcine hemoglobin. Another seal portal vessels (19). Dopamine appears to be a major aliquot of this fraction was subjected to electrospray ioniza- inhibitory agent in vivo, as it suppresses prolactin levels under tion mass spectrometric analysis to determine the size of the a variety ofphysiological conditions (20). A dopamine agonist, fragment. Signals corresponding to six peptides were ob- the ergot alkaloid bromocryptine, has been widely used clin- served. The identities of these peaks were based on the ically to suppress prolactin in patients with conditions asso- N-terminal sequences obtained above. A minor peak, Mr ciated with high blood levels of prolactin (8). 7840.6 + 2.6, corresponds to POMC residues 1-70 (theoret- In addition to catecholamines and y-aminobutyric acid, ical Mr, 7842.7) and a second peak, Mr 7727.1 ± 6.3, peptidic PIFs have been demonstrated by various investiga- corresponds to residues POMC 1-68 (theoretical Mr, 7728.6). tors in hypothalamic extracts (9-13). SS-14 was observed to These masses indicate that the four cysteine residues have have PIF activity under certain experimental conditions (21, formed two disulfide bonds and that the N-linked glycosyl- 22). GAP was found to suppress prolactin release in some in ation site contains no carbohydrate. Major peaks at Mr 7854.6 vitro tests (16, 23, 24). GAP also had modest prolactin + 2.4 and 7740.1 ± 2.3 correspond to POMC residues 1-70 release-inhibiting activity in some in vivo systems associated and 1-68, respectively, with a modification, most likely with high, stimulated prolactin levels (25), but it did not methylation (adds 14 u). Other modifications, such as hy- inhibit basal levels of prolactin (26). This peptide was also droxylation or methionine oxidation (adds 16 u), although inactive in the pituitary superfusion system (26). possible, lie at the extremes for expected mass accuracy Because of the controversy on possible peptide inhibitors (±0.02%). One minor peak at Mr 7206.7 + 0.6 corresponded of prolactin release, we attempted to isolate such peptides to porcine hemoglobin a chain residues 1-69 (theoretical Mr, from hypothalamic extracts by modern separation tech- 7207.2). Another minor peak ofMr 7452.2 ± 3.8 did not match niques, monitoring fractions with a sensitive pituitary cell any sequences of POMC or hemoglobin (assuming the N-ter- culture assay and determining somatostatin and GAP levels minal sequences found earlier). of the fractions by RIA. Fraction JGG-7-169 no. 10 was subjected to protein se- As a result of this isolation procedure, we purified two quencing. This preparation also gave two main sequences. fractions with high in vitro prolactin release-inhibiting activ- The dominant sequence was Ala-Ser-Asp-Arg-Ser-Asn-Ala- ity. Both compounds were peptides as based on sequence Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu-Arg- determination by Edman degradation. Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro- One of the peptides was found to be the N-terminal Ala-Gln-Pro-Gly-Val-Tyr. This sequence is identical to the fragment of the POMC precursor protein (27, 28). Synthetic residues 109-147 peptide region of porcine vasopressin- peptides corresponding to this region of POMC-namely, neurophysin, whereas the sequence present in a lesser residues 1-28, 1-36 (data not shown), and 1-49-were also amount corresponded to an internal region of the porcine found to have significant in vitro PIF activity, comparable to hemoglobin a chain. that of the natural molecule (Table 3). However, none of the Downloaded by guest on September 29, 2021 3544 Medical Sciences: Schally et al. Proc. Natl. Acad. Sci. USA 88 (1991) peptides inhibited prolactin secretion in a pituitary superfu- cologic Endocrinology and Infertility (Williams & Wilkins, sion assay (data not shown). It is interesting to note that Baltimore), 3rd Ed., p. 589. (30) have 9. Enjalbert, A., Moos, F., Carbonell, L., Priam, M. & Kordon, interleukin 1 (29) and corticotropin-releasing factor C. (1977) Neuroendocrinology 24, 147-161. also been found to inhibit prolactin release in vitro. Both 10. Greibrokk, T., Hansen, J., Knudsen, R., Lam, Y.-K. & Folk- molecules stimulate the release of POMC-derived peptides, ers, K. (1975) Biochem. Biophys. Res. Commun. 67, 338-344. including corticotropin and f3-endorphin, as well as the N-ter- 11. Mizunuma, H., Khorram, 0. & McCann, S. M. (1985) Proc. minal fragment. It is tempting to speculate that the N-terminal Soc. Exp. Biol. Med. 178, 114-120. peptide of POMG may be a mediator of the observed effects 12. Khorram, O., DePalatis, L. R. & McCann, S. M. (1983) Annu. Meet. Endocr. Soc. 6Sth, 1983, p. 233 (abstr. 612). of interleukin 1. 13. Schally, A. V., Coy, D. H. & Meyers, C. A. (1978) Annu. Rev. The other peptide obtained from the purification was found Biochem. 47, 89-128. to be a fragment (copeptin) of the vasopressin-neurophysin 14. Esch, F., Bohlen, P., Ling, N., Brazeau, P. & Guillemin, R. precursor (31). This peptide appeared similar to the glyco- (1983) Biochem. Biophys. Res. Commun. 117, 772-779. peptide previously isolated from pig, ox, and sheep pituitaries 15. Arimura, A., Lundqvist, G., Rothman, J., Chang, R., Fernan- (32) and also characterized in other mammalian species (31). dez-Durango, R., Elde, R., Coy, D. H., Meyers, C. & Schally, Nagy et al. (33) reported that this 39-amino acid glycopeptide A. V. (1978) Metabolism 27, Suppl. 1, 1139-1144. 16. Nikolics, K., Mason, A. J., Szonyi, E., Ramachandran, J. & comprising the C-terminal of the vasopressin-neurophysin Seeburg, P. H. (1985) Nature (London) 316, 511-517. precursor stimulated prolactin release in vitro. The discrep- 17. Henzel, W. J., Rodriguez, H. & Watanabe, C. (1985) J. Chro- ancy between their results (33) and ours can be explained at matogr. 404, 41-52. present only by differences in the assays used. Future in vivo 18. Hokfelt, T. (1967) Brain Res. 5, 121-123. studies can demonstrate whether the substances we isolated, 19. Plotsky, P. M., Gibbs, D. M. & Neil, J. D. (1978) Endocrinol- or their congeners, may play a physiological role in prolactin ogy 102, 1887-1894. release. 20. Neill, J. D. (1980) in Frontiers in Neuroendocrinology, eds. Martini, L. & Ganong, W. F. (Raven, New York), pp. 129-155. 21. Vale, W. W., Rivier, C., Brazeau, P. & Guillemin, R. (1974) We are grateful to Dr. H. P. J. Bennett for the gift ofbovine POMC Endocrinology 98, 968-977. fragments and Dr. R. Acher and Dr. J. Chauvet for sheep MSEL- 22. McCann, S. M. (1982) Annu. Rev. Pharmacol. Toxicol. 22, neurophysin. The participation in this project of Dr. V. Csernus, 491-515. Weldon Carter, and Don Olson is gratefully acknowledged. We are 23. Collu, R., Forget, H. & Lafond, J. (1988) Annu. Meet. Endocr. grateful to Karl Clauser for his help with mass spectrometric analysis Soc. 70th, 1988, p. 66 (abstr. 182). and to Mark Nixon for the synthetic POMC fragment (residues 1-49). 24. Wormald, P. J., Abrhamson, M. J., Seeburg, P. H., Nikolics, We thank the National Hormone and Pituitary Program (National K. & Millar, R. P. (1989) Clin. Endocrinol. 30, 149-155. Institute of Diabetes and Digestive and Kidney Diseases) for gifts of 25. Yu, W. H., Seeburg, P. H., Nikolics, K. & McCann, S. M. materials used in RIAs. This work was supported by National (1988) Endocrinology 123, 390-395. Institutes ofHealth Grant AM 07467 and the Department ofVeterans 26. Schally, A. V., Olsen, D. B., Gulyas, J., Szoke, B., Horvath, Affairs Research Service to A.V.S. J., Karashima, T., Redding, T. W., Nikolics, K. & Seeburg, P. H. (1986) Annu. Meet. Endocr. Soc. 68th, 1986, p. 26 (abstr. 1. Pasteels, J. C. (1962) C. R. Acad. Sci. Ser. 2 254, 2664-2666. 8). 2. Talwalker, P. 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