Proc. Nadl. Acad. Sci. USAVol. 88, pp. 3540-3544, May 1991Medical Sciences

Isolation and characterization of two peptides with prolactinrelease-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 ofExperimental Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112; and tDepartments of DevelopmentalBiology 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-inhibiting activity were purified from stalk median eminence(SME) fragments of 20,000 pig hypothalami. Monolayer cul-tures ofrat anterior pituitary cells were incubated with aliquotsof chromatographic fractions and the inhibition of release ofprolactin in vitro was measured by RIA in order to monitor thepurification. The hypothalamic tissue extract was separatedinto 11 fractions by high-performance aqueous size-exclusionchromatography with one fraction showing a 4-fold increase inprolactin release-inhibiting factor (PIF) activity. This materialwas further purified by semipreparative reversed-phase (RP)HPLC. This process resulted in the separation of two distinctfractions that showed high PIF activity. These were furtherpurified by semipreparative and analytical RP-HPLC to ap-parent homogeneity as judged by the UV absorbance profiles.Neither of the two peptides showed cross-reactivity with go-nadotropin releasing hormone-associated peptide or with so-matostatin-14 antibodies. Protein sequence analysis revealedthat one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-Ala-Cys-Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 ofthe N-terminal region of the proopiomelanocortin (POMC)precursor (corresponding to amino acids 1-26 of the 16-kDafragment). The sequence of the other PIF was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu-Arg-Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr, representing res-idues 109-147 of the vasopressin-neurophysin precursor.Synthetic peptides corresponding to the N-terminal region ofPOMC had significant PIF activity in vitro.

The existence of prolactin release-inhibiting factor(s) (PIF) inrat hypothalamic extracts was first demonstrated by Pasteels(1) and Talwalker et al. (2). Several substances were lateridentified in mammalian hypothalamic tissues that inhibitedthe release of prolactin (3-7). In humans, the administrationof dopamine receptor antagonists, such as chlorpromazineand other neuroleptics, markedly increases prolactin level,whereas dopamine agonists, such as the ergot derivatives,significantly reduce plasma prolactin concentration (8).Based on these clinical and experimental data, dopamine wasconsidered to be the only physiological PIF.On the other hand, peptidic substances have been partially

purified from brain extracts, which also had significant PIFactivity (9-12). Our preliminary results also suggested thatdopamine is not the only hypothalamic substance with PIFactivity (13). However, the isolation of a highly potentpeptide PIF from hypothalamic extracts has not yet beenaccomplished.

Here we report the isolation of two peptides from porcinehypothalami, different from gonadotropin releasing hor-mone-associated peptide (GAP) and somatostatin-14 (SS-14),that exhibited a dose-dependent prolactin release-inhibitingactivity in vitro. These substances or their congeners mightplay a physiological role in the regulation ofprolactin release.

MATERIALS AND METHODSIsolation of the two peptides with PIF activity from 20,000lyophilized stalk median eminence (SME) fragments of pighypothalami (Oscar Mayer, Madison, WI) was accomplishedessentially by sequential purification in six steps. After eachpurification step, the fractions were pooled and their in vitroPIF activity and levels of immunoreactive GAP and SS-14were determined.

Extraction. Extraction was carried out as described indetail by Schally et al. (3). Briefly, lyophilized fragments of20,000 pig hypothalami, weighing 531 g, were first pulverized,defatted by acetone and petroleum ether, extracted with 2 Macetic acid at 80C, and centrifuged (3). Phenylmethylsulfonylfluoride and pepstatin A (10 pug/ml each) were added to theclear supernatant (14). The mixture was heated to boiling,immediately cooled on ice to 40C, and centrifuged. The clearsupernatant was lyophilized, resulting in 114.5 g of dryextract from porcine hypothalami.

Preparative Size-Exdusion HPLC. From the lyophilizedhypothalamic extract, 98 g (16,200 SME) was dissolved in50% acetic acid (71.5 ml), diluted with distilled water to 4290ml (final pH 2), and centrifuged (Sorvall RC S B; 26,890 x g;30 min). The clear supernatant was then subjected to high-performance aqueous size-exclusion chromatography on aTSK G-2000SW (21.4 x 600 mm) column (Toyo-Soda, Phe-nomenex, Rancho Palos Verdes, CA), after equilibration ofthe stationary phase with 10 bed vol of 0.1 M NaCl/0.05 MTris'HC1, pH 4.4. The flow rate of the mobile phase was 6ml/min. In total, 88 separate chromatographic runs wereperformed under identical conditions. In each run, 11 frac-tions were collected (Fig. la).

Preparative Reversed-Phae (RP) HPLC. Fraction JGG-2-53 no. 3/1 in aliquots of 90 ml from the TSK G-2000SWcolumn, exhibiting significant in vitro PIF and immunoreac-tive GAP activities, was subjected to RP chromatography ona Dynamax C18 preparative column (Rainin Woburn, MA)(250 x 41.4 mm; 12-pum particle size; 300-X pore size). Alinear gradient was used for the elution of the substancesadsorbed to the matrix of the column. Component B of themobile phase was increased from 0% to 10%o in 70 min

Abbreviations: PIF, prolactin release-inhibiting factor; POMC, proo-piomelanocortin; GAP, gonadotropin releasing hormone-associatedpeptide; RP, reversed-phase; SS-14, somatostatin-14; SME, stalkmedian eminence.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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g

0 40 801.0o iiri

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10Time, min

FIG. 1. Chromatographic purification of the two peptides withprolactin release-inhibitory activity. Extracts of 20,000 porcine hy-pothalami were chromatographed in successive steps by size-exclusion HPLC (a) and preparative RP-HPLC (b). The shaded ordarkened areas represent fractions with the highest in vitro prolactinrelease-inhibitory activity that were collected and processed insubsequent steps. Fraction 12 of b (JGG-2-127) was further chro-matographed by preparative and analytical RP-HPLC (c-f). Fraction13 of b was further chromatographed by RP-HPLC (g-i). The shadedareas of f (JGG-7-29-12) and darkened areas of i (JGG-7-169-10),respectively, were analyzed by protein sequencing and mass spec-trometry.

(solvent A, 0.1% aqueous trifluoroacetic acid; solvent B,0.1% aqueous trifluoroacetic acid in 70o acetonitrile), whileusing a flow rate of 62 ml/min (Fig. lb). Sixteen fractions(JGG-2-127) were collected and pooled from each of the 16separate runs.

Purification Scheme A. One of the two fractions with thehighest in vitro PIF activity (JGG-2-127 no. 12), weighing 100mg, was dissolved in 50%o solvent A/50% solvent B andfiltered through a hydrophilic, chemically resistant, 25-mmnylon Acrodisc filter unit (0.2-pum pore size) (Gelman). It wasthen subjected to RP chromatography on a Dynamax C18(Rainin) (250 x 21.4 mm; 12-,um pore size; 300-A particlesize) preparative column. The same mobile phase was usedas in the previous preparative RP separation step, with adifferent gradient. Component B of the mobile phase was

increased from 35% to 70% in 140 min, while using a flow rateof 8 ml/min (Fig. 1c). Twenty-four fractions (JGG-2-149)were collected and Iyophilized. A 16-mg aliquot of thematerial with the highest in vitro PIF activity (JGG-2-149 no.14) was further purified on a Vydac C18 (Rainin) 250 X 10.0mm, 5-pum particle size and 300-A pore size semipreparativeRP column, using the same mobile phase as in preparativeRP-HPLC. A flow rate of 2 ml/min and a shallow lineargradient was used. Component B of the mobile phase wasincreased from 40% to 62% in 72 min (Fig. ld). Twenty-fivefractions were collected and Iyophilized (JGG-2-189).A 6-mg fraction with the highest PIF activity (JGG-2-189

nos. 17 and 18) was rechromatographed on the same VydacC18 RP column as in step III using the trifluoroacetic acid/acetonitrile/water, mobile phase, with a shallow linear gra-dient, increasing component B of the mobile phase from 35%to 70% in 140 min (Fig. le). The separation resulted in 27fractions (JGG-7-29).The most potent PIF fraction (JGG-7-29 no. 12), weighing

71 pAg, was subjected to purity tests on an Aquapore RP-300(Brownlee, Phenomenex), 250 x 1.0 mm, 7-pum, 300-A mi-crobore column, using a linear gradient (solvent A, 0.1%trifluoroacetic acid/water; solvent B, 0.1% trifluoroaceticacid/70% acetonitrile/30%o water) (Fig. if), with,a flow rateof 0.08 ml/min. Component B of the mobile phase wasincreased from 40% to 52% in 40 min. The fraction wassubjected to protein sequencing.

Purification Scheme B. The second fraction from prepara-tive RP-HPLC with high in vitro PIF activity (JGG-2-127 no.13) was dissolved in 50%o solvent A/50% solvent B, filteredthrough a hydrophilic, chemically resistant, 25-mm nylonAcrodisc filter unit (0.2-pmm pore size) (Gelman) and sub-jected to repeated separation on the same preparative columnand in the same mobile phase as described in the previous RPseparation step. However, the linear gradient was designed tobe shallow. Component B of the mobile phase was increasedfrom 40% to 62% in 65 min. Sixty fractions were collected andlyophilized (JGG-2-219) (Fig. ig).

Fraction JGG-2-219 no. 30 with the highest in vitro PIFactivity was further purified on a W-Porex 5C18 (Phenom-enex) (250 x 4.6 mm; 5-,um particle size; 300-A pore size)analytical RP column, using the same mobile phase as inpreparative RP separation. A flow rate of 1.2 ml/min and ashallow, linear gradient was used. Component B of themobile phase was increased from 42% to 64% in 40 min.Fifteen fractions were collected and Iyophilized (JGG-7-169)(Fig. lh).The most potent fractions in the PIF bioassay, JGG-7-169

no. 10, weighing 200 pLg, was subjected to a purity test onAquapore RP-300 under the same conditions as in scheme A(Fig. ii).

All the separation procedures were performed at roomtemperature, but the fractions were collected and kept at 40C.The UV absorbance of the fractions eluted from the columnswas measured at 220 and 280 nm. For the preparativepurification process, a Beckman HPLC system (Beckman,Berkeley, CA) with a 450 data system controller, two 114 Msolvent delivery modules, a 340 organizer, a 165 variablewavelength detector, a Kipp and Zonen BD41 recorder, anda Gilson model 201 fraction collector or a MacRabbit HPLCsystem (Rainin), with a Knauer UV photometer, a Gilsonmodel 202 fraction collector, a Kipp and Zonen BD41 re-corder were used. For the analytical steps, a HP-1090 liquidchromatograph HPLC system (Hewlett-Packard) was used.The solvents were HPLC grade, and the reagents were

HPLC or analytical grade in purity, obtained from Burdickand Jackson, Sigma, and from Fluka. Distilled water was alsopurified through a Milli-Q water purification system (Milli-pore).

IN 0.5.

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Prolactin and SS-14 RIA. RIA for prolactin was carried outwith materials supplied by the National Hormone and Pitu-itary Program (National Institute of Diabetes and Digestiveand Kidney Diseases). The prolactin concentrations in thesamples were measured in duplicate by a double antibodyRIA method, using as standard rat prolactin, the RP3 refer-ence preparation. The statistical significance was assessed byDuncan's new multiple range test. For the RIA of SS-14 (15)JH 204 antibody was used.GAP RIA. Substances with GAP immunoreactivity were

determined by a RIA method, using the KN-16 antibody in a1: 40,000 final dilution (16). The iodination of the antigen wasperformed by the chloramine-T method. The labeled hor-mone was repurified by gel filtration on a Sephadex G-50column. The specific activity of the labeled hormone was1375 ,uCi/,ug (1 Ci = 37 GBq). The standard curve was set upin the range between 0.10 pg/1lt and 1 ng/,pi. The binding ofthe labeled hormone to the antibody was 28%.' The interassayand intraassay variations were less than 12% and 10%,respectively.PIF in Vitro Bioassay. The monolayer culture assay will be

reported in detail elsewhere. Briefly, anterior pituitaries fromdonor female rats weighing 200-250 g were removed asepti-cally and dispersed with 0.3% collagenase in Dispase (50units/ml) and DNase (10 pug/ml). Cells were washed twiceand 0.3 x 106 cells were plated per well in 24-well culturedishes in Dulbecco's modified Eagle's medium (DMEM) plus10% fetal calf serum. Cells were incubated at 370C in 5%C02/95% air for 5 days before they were used in the assaysystem. On the day of the assay, cultures were washed twicewith DMEM without serum. Samples dissolved in this samemedium were added as 1-ml aliquots to each of four wells.Cultures were incubated an additional 24 hr and aliquots ofthe medium were taken after 4 and 24 hr for determination ofprolactin levels by RIA. PIF activity was arbitrarily definedas the prolactin release-inhibiting activity ofthe compound ascompared to the effect of the control material in the samebioassay. The effect was expressed as the percentage ofactivity of the control material in units per SME or units/mg.Since catecholamines and SS-14 contribute to PIF activity inhypothalamic extracts in this assay, the PIF activity in unitsper SME was at times decreased after a purification step dueto the elimination of these substances.

Protein Sequencing. Purified fractions of the final chro-matographic runs were analyzed by N-terminal protein se-quencing. Automated Edman degradation was performedwith an Applied Biosystems model 470A gas-phase se-quencer equipped with a 120A phenylthiohydantoin aminoacid analyzer. Phenylthiohydantoin-derivatized amino acidswere identified by RP-HPLC and integrated with a Nelsonanalytical model 3000 data system. Sequence interpretationwas performed on a Vax 11/785 computer (Digital Equip-ment) as described (17).

Electrospray Ionization Mass Spectrometry. Electrosprayionization mass spectra were obtained with a Sciex API IIItriple quadrupole mass spectrometer (Thornhill, Ontario,Canada), using a pneumatically assisted Ionspray nebulizer.The peptide was dissolved in 10% formic acid with water/acetonitrile (1:1), and it was pumped into the nebulizer (5000V) at 5 1L/min. The orifice was maintained at 120 V. The massaxis was scanned from 600 to 1600 units (u) in 15 sec using0.2-u steps.

RESULTS

The 2 M acetic acid extraction of 20,000 pig hypothalamiresulted in a yield of 114.5 g of dry powder. For isolation offractions with PIF activity, 98 g of this material was subjectedto purification by various HPLC methods (Table 1 andTable 2).

Table 1. HPLC purification scheme for a porcine hypothalamicpeptide (JGG-7-29 no. 12) with PIF activity in pituitarycell culture

Prolactin

Total SS-14, GAP, release*Sample wt pg/SME ng/SME 4 hr 24 hr

20,000 pighypothalami 531 g

AVS-10-122 114.5 g2 M acetic acid

extractionJGG-2-53 no. 3/1 2.81 37 74JGG-2-127 no. 12 100 mg 12,278 1.86 41 51JGG-2-149 no. 14 16 mg 1,487 1.24 37 49JGG-2-189 nos. 17 + 18 1 mg 5,041 18.7 21 33JGG-7-29 no. 12 131 ,ug 0 0 58 39The duration of the incubation of the cells with the tested materials

was 4 and 24 hr in each experiment.*Decrease in prolactin release, % control.

The TSK preparative size-exclusion HPLC concentratedPIF activity into 11 fractions. JGG-2-53 no. 3/1 was found tobe one of the most potent compounds showing 74 units ofPIFactivity per SME and 576 units ofPIF specific activity per mg,in vitro. This heterogenous fraction was concentrated andseparated on a preparative C18 reversed-phase column. Theprocess resulted in three fractions with significant PIF activ-ity. The greatest biological activity was found in the 100-mgfraction designated JGG-2-127 no. 12, with 51 units of PIFactivity per SME or 2040 units ofPIF activity per mg in vitro.The chromatography performed on a C18 column using ashallow gradient (0.25% solvent B increase per min) producedfraction JGG-2-149 no. 14, weighing 16 mg. This fractionshowed 49 units of PIF activity per SME or 6125 units of PIFactivity per mg and was still heterogenous (Table 1).

After purification on another C18 column, two fractions(JGG-2-189 nos. 17 and 18) were obtained (Fig. ld), weighing1 mg and exhibiting 33 units ofPIF activity per SME or 16,500units of PIF activity per mg. Fraction JGG-2-189 no. 17exhibited a dose-dependent PIF effect in vitro (Table 1) butshowed significant cross-immunoreactivity with SS-14 andGAP in the RIA (Table 1).The final analytical chromatography using acetonitrile/

trifluoroacetic acid/water, mobile phase, with a shallowgradient (0.25% solvent B increase per min) resulted in 71 ,ugof fraction JGG-7-29 no. 12. An estimated 59.5 ,ug of thismaterial was used up for the RIAs and bioassays to guide thepurification. Considering all these data, the yield of theHPLC purification ofJGG-7-29 no. 12 was 130.5 ,ug or 91.2%.The isolated material exhibited a strong UV absorption at 220nm and a lesser one at 280 nm and showed 93% homogeneityas assessed by UV absorbance (Fig. 1f) and had a PIFactivity estimated at 39 units per SME or 55,715 units/mg.JGG-7-29 no. 12 was found to be the most potent PIF fractionin the 24-hr incubation test ofthe pituitary cells. This material

Table 2. HPLC purification scheme B of a porcine hypothalamicpeptide (JGG-7-169 no. 10) with PIF activity

Prolactin

Total SS-14, GAP, release*Sample wt pg/SME ng/SME 4 hr 24 hr

JGG-2-127 no. 13 1094 0.65 35 30JGG-2-219 no. 30 810 5.30 4 27JGG-7-169 no. 10 240 ,ug 0 0 13 30

, Not available. Incubation periods of4 and 24 hr in pituitary cellculture were used for the bioassay.*Decrease in prolactin release, % control.

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exhibited no cross-reactivity in the RIAs for SS-14 or GAP.This chromatographic procedure resulted in a 96.7-fold in-crease in PIF activity when assayed in the pituitary cellculture system (Table 1). This fraction was subjected tostructural analysis.

Fraction JGG-2-127 no. 13 with high PIF activity waspurified separately according to scheme B (Fig. 1 g-i). Thechromatography performed on a Dynamax C18 column butusing a shallow gradient (0.25% solvent B increase per min)resulted in JGG-2-219 no. 30 as the most potent fraction. Thefinal semipreparative chromatography, using acetonitrile/trifluoroacetic acid/water, mobile phase, but shallow gradi-ent (0.25% solvent B increase per min) on a W-Porex C18column, results in 240 Ag of JGG-7-169 no. 10. An estimated40 pug of this substance was used for the RIAs and bioassaysto guide the purification procedures. Thus, the yield of theHPLC purification of JGG-7-29 no. 12 was 91.2%. Theisolated substance exhibited strong UV absorption at 220 nm,no UV absorption at 254 nm, and low absorption at 280 nm,and a PIF activity of 17,391 units/mg. JGG-7-169 no. 10 wasfound to be a potent fraction showing PIF activity after a 4-and a 24-hr incubation period in the pituitary cell culturebioassay. The material showed no cross-reactivity to SS-14or GAP by RIA.

This chromatographic procedure resulted in a 30.1-foldincrease in PIF specific activity in the pituitary cell culturesystem (Table 2). Fraction JGG-7-169 no. 10 was subjected toprotein sequence analysis by Edman degradation.

Protein Sequencing and Mass Spectrometric Analysis. Analiquot of fraction JGG-7-29 no. 12 was subjected to proteinsequencing for 26 cycles. The sample was found to contain asthe highest signal Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu-Ala-Cys-Ile-Arg-Ala-Cys-Lys-Pro. The sequence corresponds to residues27-52 of the N-terminal region of the porcine proopiomel-anocortin (POMG) peptide and residues 1-26 of the 16-kDafragment. Underlying this sequence, another signal wasdetected in every cycle, which corresponded to the N-ter-minal region of the a chain of porcine hemoglobin. Anotheraliquot of this fraction was subjected to electrospray ioniza-tion mass spectrometric analysis to determine the size of thefragment. Signals corresponding to six peptides were ob-served. The identities of these peaks were based on theN-terminal sequences obtained above. A minor peak, Mr7840.6 + 2.6, corresponds to POMC residues 1-70 (theoret-ical Mr, 7842.7) and a second peak, Mr 7727.1 ± 6.3,corresponds to residues POMC 1-68 (theoretical Mr, 7728.6).These masses indicate that the four cysteine residues haveformed two disulfide bonds and that the N-linked glycosyl-ation site contains no carbohydrate. Major peaks at Mr 7854.6+ 2.4 and 7740.1 ± 2.3 correspond to POMC residues 1-70and 1-68, respectively, with a modification, most likelymethylation (adds 14 u). Other modifications, such as hy-droxylation or methionine oxidation (adds 16 u), althoughpossible, lie at the extremes for expected mass accuracy(±0.02%). One minor peak at Mr 7206.7 + 0.6 correspondedto porcine hemoglobin a chain residues 1-69 (theoretical Mr,7207.2). Another minor peak ofMr 7452.2 ± 3.8 did not matchany sequences ofPOMC or hemoglobin (assuming the N-ter-minal sequences found earlier).

Fraction JGG-7-169 no. 10 was subjected to protein se-quencing. This preparation also gave two main sequences.The dominant sequence was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu-Arg-Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val-Tyr. This sequence is identical to theresidues 109-147 peptide region of porcine vasopressin-neurophysin, whereas the sequence present in a lesseramount corresponded to an internal region of the porcinehemoglobin a chain.

Table 3. PIF activity of purified hypothalamic PIF and rat,bovine, and human POMC fragments in the pituitary cellculture bioassay

Prolactin

Dose,* ng/ml releasetSample ,ug At 4 hr At 24 hr 4 hr 24 hr

Control - 1730 ± 70 11,413 ± 400 -JGG-2-189

no. 17 0.1 1330 ± 60 7,800 ± 600 23f 32tJGG-2-189

no. 17 0.5 1187 ± 170 4,075 ± 600 31§ 64§Control - 1040 ± 22 5,850 ± 150 -Rat 0.01 1000 ± 26 4,630 ± 88 4 21§POMC 0.1 920 ± 19 3,730 ± 312 11t 36§

1-49 1.0 790 ± 27 3,230 ± 78 24§ 45§Bovine 0.01 930 ± 17 4,580 ± 287 11t 22§POMC 0.1 960 ± 14 4,950 ± 215 8 15t

1-49 1.0 800 ± 38 4,240 ± 317 23§ 28§Human 0.01 740 ± 64 4,200 ± 235 29§ 28§POMC 0.10 920 ± 96 4,280 ± 127 11t 27§

1-49 1.0 800 ± 10 4,690 ± 120 23§ 20§*Dry weight.tDecrease in prolactin secretion release, % control.tSignificantly different from control; P < 0.05.§Significantly different from control; P < 0.01.

DISCUSSIONThe hypothalamic control of pituitary prolactin release ismediated by stimulatory and inhibitory agents (8). Severalcompounds are known to inhibit prolactin release in pharma-cological doses, such as dopamine, norepinephrine, y-ami-nobutyric acid, and acetylcholine (13). Purification of variousfractions with PIF activity from hypothalamic extracts in ourlaboratory led to the isolation ofdopamine and norepinephrine(6), as well as y-aminobutyric acid (7). Dopamine is present ina high concentration in median eminence (18) and in hypophy-seal portal vessels (19). Dopamine appears to be a majorinhibitory agent in vivo, as it suppresses prolactin levels undera variety ofphysiological conditions (20). A dopamine agonist,the ergot alkaloid bromocryptine, has been widely used clin-ically to suppress prolactin in patients with conditions asso-ciated with high blood levels of prolactin (8).

In addition to catecholamines and y-aminobutyric acid,peptidic PIFs have been demonstrated by various investiga-tors in hypothalamic extracts (9-13). SS-14 was observed tohave PIF activity under certain experimental conditions (21,22). GAP was found to suppress prolactin release in some invitro tests (16, 23, 24). GAP also had modest prolactinrelease-inhibiting activity in some in vivo systems associatedwith high, stimulated prolactin levels (25), but it did notinhibit basal levels of prolactin (26). This peptide was alsoinactive in the pituitary superfusion system (26).Because of the controversy on possible peptide inhibitors

of prolactin release, we attempted to isolate such peptidesfrom hypothalamic extracts by modern separation tech-niques, monitoring fractions with a sensitive pituitary cellculture assay and determining somatostatin and GAP levelsof the fractions by RIA.As a result of this isolation procedure, we purified two

fractions with high in vitro prolactin release-inhibiting activ-ity. Both compounds were peptides as based on sequencedetermination by Edman degradation.One of the peptides was found to be the N-terminal

fragment of the POMC precursor protein (27, 28). Syntheticpeptides corresponding to this region of POMC-namely,residues 1-28, 1-36 (data not shown), and 1-49-were alsofound to have significant in vitro PIF activity, comparable tothat of the natural molecule (Table 3). However, none of the

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peptides inhibited prolactin secretion in a pituitary superfu-sion assay (data not shown). It is interesting to note thatinterleukin 1 (29) and corticotropin-releasing factor (30) havealso been found to inhibit prolactin release in vitro. Bothmolecules stimulate the release of POMC-derived peptides,including corticotropin and f3-endorphin, as well as the N-ter-minal fragment. It is tempting to speculate that the N-terminalpeptide ofPOMG may be a mediator of the observed effectsof interleukin 1.The other peptide obtained from the purification was found

to be a fragment (copeptin) of the vasopressin-neurophysinprecursor (31). This peptide appeared similar to the glyco-peptide previously isolated from pig, ox, and sheep pituitaries(32) and also characterized in other mammalian species (31).Nagy et al. (33) reported that this 39-amino acid glycopeptidecomprising the C-terminal of the vasopressin-neurophysinprecursor stimulated prolactin release in vitro. The discrep-ancy between their results (33) and ours can be explained atpresent only by differences in the assays used. Future in vivostudies can demonstrate whether the substances we isolated,or their congeners, may play a physiological role in prolactinrelease.

We are grateful to Dr. H. P. J. Bennett for the gift ofbovine POMCfragments and Dr. R. Acher and Dr. J. Chauvet for sheep MSEL-neurophysin. The participation in this project of Dr. V. Csernus,Weldon Carter, and Don Olson is gratefully acknowledged. We aregrateful to Karl Clauser for his help with mass spectrometric analysisand to Mark Nixon for the synthetic POMC fragment (residues 1-49).We thank the National Hormone and Pituitary Program (NationalInstitute of Diabetes and Digestive and Kidney Diseases) for gifts ofmaterials used in RIAs. This work was supported by NationalInstitutes of Health GrantAM 07467 and the Department ofVeteransAffairs Research Service to A.V.S.

1. Pasteels, J. C. (1962) C. R. Acad. Sci. Ser. 2 254, 2664-2666.2. Talwalker, P. K., Ratner, A. & Meites, J. (1963) Am. J.

Physiol. 205, 213-218.3. Schally, A. V., Kuroshima, A., Ishida, Y., Redding, T. W. &

Bowers, C. Y. (1965) Proc. Soc. Exp. Biol. Med. 118, 350-352.4. Meites, J. & Clemens, J. A. (1972) Vitam. Horm. 30, 165-221.5. Takahara, J., Arimura, A. & Schally, A. V. (1974) Endocrinol-

ogy 95, 462-465.6. Schally, A. V., Dupont, A., Arimura, A., Takahara, J., Red-

ding, T. W., Clemens, R. & Shaar, C. (1976) Acta. Endocrinol.(Copenhagen) 82, 1-14.

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