Proc. Natl. Acad. Sci. USA Vol. 74, No. 12, pp. 5300-5304, December 1977 Biochemistry Characterization of a common precursor to corticotropin and fl-lipotropin: Identification of f-lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system* (polysome runoff peptides/immunoprecipitation) JAMES L. ROBERTSt AND EDWARD HERBERT Department of Chemistry, University of Oregon, Eugene, Oregon 97403 Communicated by George Streisinger, September 1, 1977 ABSTRACT Radioactive proteins synthesized in an ,B-endorphin is present in Mr 31,000 ACTH in AtT-20/D16v mRNA-dependent reticulocyte cell-free system under the di- cells by tryptic peptide analysis, and Giagnoni et al. (10) have rection of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either shown that AtT-20/D-1 cells contain several species of peptides a(1-24) corticotropin or f-endorphin [#(61-91) lipotropin]. Each with opiate activity. immunoprecipitate was fractionated by sodium dodecyl sul- In this study we have used tryptic peptide analysis to show fate/polyacrylamide gel electrophoresis and shown to contain that several ,B-LPH peptides are present in the cell-free product only one labeled protein with an apparent molecular weight of isolated with antisera specific to ACTH and fl-endorphin. These 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin approaches have also yielded information about the location and fl-lipotropin molecules isolated from the gels demonstrated to in the cell-free product. that the two proteins had the same lysine, methionine, and of ,B-LPH relative a(1-39) ACTH tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromato- MATERIALS AND METHODS graphic mobilities as marker tryptic peptides from bovine ,3- RNA and Polysome Isolation and Translation in a Retic- melanotropin and porcine (3-endorphin. The identification of ulocyte Cell-Free System. Membrane-bound polysomes were these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The jP-lipotropin isolated from AtT-20/D-16v tumor cells as described (1). RNA tryptic peptides were also shown to be located carboxy terminal was extracted from these polysomes with phenol/chloroform to the corticotropin tryptic peptides. and precipitated with LiCl (11). RNA and polysomes were translated in an mRNA-dependent reticulocyte system as de- When messenger RNA from cultures of mouse pituitary cells scribed (1, 12). Aurintricarboxylic acid was used to inhibit (AtT-20/D-16v cell line) that secrete corticotropin (ACTH) is initiation of protein synthesis in the polysome chain completion translated in a reticulocyte cell-free system, a species of ACTH experiments (13). At 0.1 mM, aurintricarboxylic acid reduced is synthesized with an apparent molecular weight (Mr) of 28,500 AtT-20 RNA-stimulated incorporation of labeled amino acid (1). The same size ACTH molecule is made when polysomes into protein to 2% of the control level. from AtT-20 cells direct the cell-free synthesis of protein (1). Immunoprecipitation and Sodium Dodecyl Sulfate/ The Mr 28,500 product contains the same methionine, lysine, Polyacrylamide Gel Electrophoresis. Immunoprecipitation and phenylalanine peptides as a class of higher molecular of the labeled lysate material was performed as previously weight ACTH glycoproteins (Mr 31,000) observed in the described (1). The ACTH antibody is specific to the ac(11-24) AtT-20 cells (2). The kinetics of labeling of these glycoproteins region of ACTH (3, 14) and the (3-endorphin antibody (RB-100) with radioactive amino acids shows that they are the precursors is specific to the fl-endorphin region of ,B-LPH (15) and reacts of the lower molecular weight forms of ACTH found in the cells equally well with (3-endorphin and fl-LPH (15). Carrier non- (Mr 23,000, 13,000, and 4500 ACTH) (3). Hence, the Mr 28,500 immune rabbit immunoglobulin was not necessary in forming cell-Iree product appears to be the primary translation product the double antibody complex with goat antiserum to rabbit from which all the forms of ACTH in the AtT-20 cells are de- immunoglobulin when the fl-endorphin antibody was used. The rived. immunoprecipitates were analyzed by BioPhore 12% sodium Tryptic peptide analysis of the cell-free product has shown dodecyl sulfate (NaDodSO4)/polyacrylamide gel electropho- that it contains one copy of a(1-39) ACTH, leaving approxi- resis (1). mately 210 amino acids unaccounted for (1). The finding that Tryptic Peptide Analysis of the Cell-Free Product. Cell-free ACTH and fl-melanotropin (f,-MSH) [,(41-58) lipotropin ACTH or (-endorphin was eluted from the NaDodSO4 gel and (LPH)] are present in the same cells in the pituitary (4) and that digested with trypsin treated with L-1-tosylamido-2-phenyl- concentrations of these hormones in plasma rise and fall to- ethyl chloromethyl ketone (TPCK), and the digest was analyzed gether under some circumstances (5-7) suggested to us that a portion of the sequence of Mr 28,500 ACTH might be Abbreviations: MSH, melanotropin, melanocyte-stimulating hormone); f3-LPH. ACTH, corticotropin (adrenocorticotropic hormone); LPH, lipotropin Interest in the synthesis of O3-LPH has intensified recently with (lipotropic hormone); NaDodSO4, sodium dodecyl sulfate; the discovery that it contains the opiate peptide f,-endorphin e-Dnp-lysine, e-dinitrophenyllysine; Mr, molecular weight; cell-free [(3(61-91) LPH] (8). Mains et al. (9) have provided evidence that ACTH or (3-endorphin, the cell-free product purified by immu- noprecipitation with antiserum to ACTH or f,-endorphin. The costs of publication of this article were defrayed in part by the * This is the second of two papers on the common precursor to corti- payment of page charges. This article must therefore be hereby marked cotropin and ,B-lipotropin. The preceding paper is ref. 1. "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Present address: Metabolic Research Unit, 1143 HSW, University of this fact. California, San Francisco, CA 94143. 5300 Downloaded by guest on September 24, 2021 Biochemistry: Roberts and Herbert Proc. Natl. Acad. Sci. USA 74 (1977) 5301 Table 1. Immunoprecipitation of the cell-free product by specific antisera for ACTH and fl-endorphin 6 % of total cpm in e " 55 immunoprecipitate with 10 antiserum to: Y 'I x 4 I I Competing peptide* ACTH f3-Endorphin E i t' I 3 None 100 100 o-~~~~~~ a(1-39) ACTH (porcine) 4 93 2 fl-Endorphin 94 7 * Each peptide was added at 10 Asg to give approximately a 1000-fold excess of unlabeled peptide over cell-free product. 10 20 30 40 50 60 mm from top of gel tides from cell-free ACTH have the same mobilities as the cell-free FIG. 1. Size analysis of ACTH and fl-endorphin synthesized in methionine peptides from fl-endorphin. Electropho- or the reticulocyte cell-free system. AtT-20 RNA was incubated in the retic profiles of the tryptic peptides of [3H]tryptophan- reticulocyte lysate system with [3H]lysine (20 ,gM, 60 Ci/mmol, Am- [3H]lysine-labeled cell-free ACTH or fl-endorphin show that ersham/Searle). The product (-10 ng) was purified by immunopre- the two proteins have very similar peptide contents (Fig. 2 B cipation using either the purified ACTH antibody or the f-endorphin and C). These data, taken together with the results of the im- was antiserum. The entire immunoprecipitate (-80 ,ug of protein) munological studies, suggest that the cell-free ACTH and f- subjected to electrophoresis on a 12% BioPhore gel, and 1-mm slices endorphin molecules are the same. For the remainder of the were cut, eluted, and analyzed for 3H radioactivity in a Triton X- 100/xylene fluor by liquid scintillation counting (efficiency -40%). experiments described below, the ACTH antibody has been Dansylated yeast alcohol dehydrogenase (Y) and dansylated soybean used to isolate the cell-free product. trypsin inhibitor (S) were included as internal markers. Molecular Identification of fl-LPH Peptides in the Cell-Free Product. weight was determined as previously described (1). --- -0, ACTH The f3-MSH and #-endorphin regions of f3-LPH are highly immunoprecipitate; 0-0, fl-endorphin immunoprecipitate. conserved (17). Hence, tryptic digests of bovine f3-MSH and porcine fl-endorphin were used as marker peptides for the by paper electrophoresis at pH 6.5 and by paper chromatog- identification of specific fl-LPH peptides in the cell-free raphy (1). Tryptic peptides of bovine ,B-MSH and porcine 13- product. The mobilities of these peptides during electrophoresis endorphin standards were visualized using Fluram (16). at pH 6.5 and during chromatograpy in butanol/acetic acid/ water are shown in Table 3. RESULTS The cell-free product was labeled with 16 different amino in the isolated with antiserum to and Size of the Cell-Free acids reticulocyte system, Immunoprecipitation Analysis ACTH, and digested with trypsin. All of the radioactive pep- Product. Tumor cell RNA was translated in a modified retic- tides were analyzed by paper electrophoresis at pH 6.5. Paper ulocyte cell-free system in the presence of radioactive amino chromatography in a second dimension was performed with acids (1). ACTH and ,B-endorphin proteins were isolated by phenylalanine-, valine-, leucine-, serine-, methionine-, and immunoprecipitating identical aliquots of labeled lysate with tryptophan-labeled peptides. The results of such an analysis antiserum to either ACTH or When these im- fl-endorphin. with [3H]phenylalanine-labeled peptides are shown in Fig. 3. munoprecipitates were analyzed by NaDodSO4 gel electro- Labeled peptides having mobilities similar to the mobilities of phoresis, only one labeled peak was observed, with an apparent the marker peptides derived from #-MSH and O-endorphin are Mr of 28,500 (Fig.