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Isolation and Characterization of Two Peptides with Prolactin Release

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Isolation and Characterization of Two Peptides with Prolactin Release 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.
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