Identification Off-Lipotropin Peptides and Their Arra

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 immunoprecipitation 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. 1). Specificity of the antisera was demon- identified in Table 3 and in Fig. 2 A and B [a(1-8) ACTH and strated by showing that excess porcine a(1-39) ACTH inhibited 1(61-69) LPH in Fig. 2A and a(9-15) ACTH and 1(52-57) binding of the cell-free product to the ACTH antiserum but not LPH in Fig. 2B]. A partial amino acid composition of these to the ,B-endorphin antiserum, and that excess ,B-endorphin peptides could be determined by observing the amount of a inhibited binding of the product to the antiserum O-endorphin particular radioactive amino acid incorporated into a particular but not to the ACTH antiserum (Table 1). peptide as described previously (1) (italicized amino acids in To determine if the two antisera were binding to the same Table 3). The profiles of the methionine- and tryptophan-la- protein, the supernatant from an immunoprecipitation carried out with the fl-endorphin antiserum was subsequently immunoprecipitated with the ACTH antiserum. Analysis of the im- Table 2. Sequential immunoprecipitations with ACTH and ,B- munoprecipitates by NaDodSO4 gel electrophoresis showed that endorphin antisera of AtT-20 RNA-directed cell-free proteins all of the Mr 28,500 cell-free product had been removed from Antibody used Total cpm in the lysate by the first immunoprecipitation (Table 2). In a Exp. First Second Mr 28,500 ACTH/LPH similar experiment using the two antisera in the reverse order, 1 ACTH 950 the same result was obtained (Table 2). 2 fl-Endorphin 910 Similarity of Tryptic Peptides in Cell-Free ACTH and 13- 3 ACTH ,B-Endorphin 15 Endorphin. To provide further evidence of the identity of the 4 f-Endorphin ACTH 18 ACTH and ,B-endorphin cell-free products, their tryptic peptides were compared. Cell-free ACTH or 13-endorphin was Two identical aliquots of labeled AtT-20 proteins were immu- purified by NaDodSO4 gel electrophoresis and digested with noprecipitated with either the ACTH or the fl-endorphin antiserum. supernatants immunoprecipitates (experiments 1 and trypsin, and the peptides were separated by paper electro- The from these 2) were then immunoprecipitated with the second antiserum (ex- phoresis and paper chromatography (1). Fig. 2A shows a two- periments 3 and 4). All four immunoprecipitates were analyzed by dimensional separation of peptides from [35S]methionine-la- NaDodSO4 gel electrophoresis and the amount of label in the Mr beled cell-free ACTH and ,B-endorphin. The methionine pep- 28,500 ACTH/LPH molecule is shown in the right-hand column. Downloaded by guest on September 24, 2021 5302 Biochemistry: Roberts and Herbert Proc. Natl. Acad. Sci. USA 74 (1977)

A 400 1- ~~~~Origin a(l - 8)- E ACTH C en .200 I-1 a'.~~~~~~~~~~~~~~~~~~~ 0.6 0.4 0.2 0 -0.2 Mobility, RLYS 400 A I ,(61 -69)- 200 ,'-LPH 4Phe E All a(-8)ACTH a 2CL200

en 0 0.5 1.0 0 05 1.0 SC/ Mobility, Rphp Phe s0 FIG. 3. Two-dimensional analysis of tryptic peptides from [3H]phenylalanine-labeled cell-free product. The cell-free product was isolated as described in Fig. 1, using the ACTH antiserum. 0 0.5 1.0 Two-dimensional peptide analysis was carried out as described in Fig. Mobility, RPhe 2. (Upper) Electrophoretic profile. (Lower) A-H, chromatographic profiles of peptides separated by electrophoresis. P indicates the position of phenylalanine.

beled peptides in Fig. 2 A and B also suggest that there is only one copy of the f-LPH sequence in the cell-free product, because the amount of radioactivity in the peptides derived from the O-LPH region of the molecule is equal to the amount of radioactivity in peptides derived from other regions of the (n molecule (1). The same kind of analysis indicates that there is only one copy of a(1-39) ACTH in the cell-free product (1). Peptide Analysis of the Product Made by Completion of Nascent Chains in Polysomes. Polysomes were incubated with labeled amino acids in the reticulocyte system in the presence of aurintricarboxylic acid at 0.1 mM to inhibit chain initiation. Because proteins are synthesized from NH2-terminus to COOH-terminus, the completed nascent chains (polysome runoff proteins) should contain a gradient of labeled amino acid, with the COOH-terminus being of the highest specific activity. The protein synthesized under the direction of mRNA will not have a gradient of label because it is made de novo. Thus, the tryptic peptides of the mRNA-directed protein will contain label of nearly equal specific activity (1), whereas the tryptic

E cm a analyses (A) duplicate samples were spotted in parallel, 2 and 5 cm in from the of a 25-cm-wide sheet of Whatman 3 MM paper. I edge E-Dnp-lysine, lysine, and picric acid were included as internal standards. Electrophoresis was done in pyridine/acetic acid/water, 10: 35:90 by volume, pH 6.5, at 2000 V/50 cm for -1.5 hr. A 3-cm strip, containing only one sample, was cut from the edge of the 25-cm sheet and sprayed with ninhydrin to locate lysine and E-Dnp lysine. The strip was cut into 8-mm slices and soaked in scintillation vials with 500 ,l of 0.1% NaDodSO4/0.5 M urea for 6 hr, 6 ml of fluor was added, and the radioactivity in the vials was measured. After the location of the labeled peptides had been determined, strips were cut from ap- Mobility, RLYS propriate regions of the sheet as indicated by the roman numerals in the upper panel in A and chromatographed in butanol/acetic acid/ FIG. 2. Similarity of tryptic peptides in cell-free ACTH and ,3- water, 4:1:5 by volume, upper phase, with phenylalanine as an internal endorphin. Cell-free product was isolated with antiserum to ACTH standard (lower three panels in A). Radioactivity was analyzed as or fl-endorphin as in Fig. 1. The protein was eluted from the gel and above. (The chromatographic profile of the origin peak is not shown precipitated with trichloroacetic acid, and the precipitate was dis- because the radioactive material did not migrate.) Electrophoretic solved in 100 Ml of 0.1 M NH4HCO3, pH 8.5. The sample was treated mobility was defined relative to lysine (e-Dnp-lysine = 0 and lysine with trypsin, lyophilized, and dissolved in 1% acetic acid. For one- = 1). Chromatographic mobility was defined relative to phenylalanine dimensional analyses ([3H]tryptophan peptides in B and [3H]lysine (RPhe = 1). Recovery of label off the paper was greater than 85% in in C) the peptides were spotted on 3-cm strips of Whatman 3 MM all cases. (A) Two-dimensional separation of [35S]methionine tryptic paper (0-0, ACTH; O...o, fl-endorphin). For two-dimensional peptides of cell-free ACTH (O--- -0) and fl-endorphin (0-0). Downloaded by guest on September 24, 2021 Biochemistry: Roberts and Herbert Proc. Natl. Acad. Sci. USA 74 (1977) 5303 Table 3. Mobilities of tryptic peptides of fl-lipotropin and the cell-free product Peptide mobilitiest Mr 28,500 ACTH/LPH Lipotropin peptide Marker peptides peptides Residues Sequence RLN RPhe RLY8 Rphe 41-46 Asp-Ser*-Gly-Pro-Tyr-Lys 0.03 0.70 0.03 0.75 47-51 Mett-Glu-His-Phe*-Arg 0.15 0.72 0.21 0.74 52-57 Trp-Gly-Ser-Pro-Pro-Lys § -§ 0.38 61-69 Tyr-Gly-Gly-Phe*-Met-Thr-Ser*-Glu-Lys 0.04 0.66 0.04 0.69 70-79 Ser*-Glu-Thr-Pro-Leu- Val*-Thr-Leu-Phe*-Lys 0.22 0.92 0.25 0.88 80-84 Asn-Ala-Ile-Ile-Lys 0.32 0.64 0.35 85-881 Asn-Ala-Tyr-Lys Tryptic peptides ofbovine #l-MSH and porcine fl-endorphin were used as marker peptides. Peptide mobilities, RLY, and Rphe, were determined as described in Fig. 2. The italicized amino acids were identified in the cell-free product tryptic peptides that had been subjected to paper electrophoresis. Amino acids used to label peptides characterized in a second dimension by chromatography are marked with an asterisk (*). t The mobilities were determined from the center ofthe spots. The average variation ofthese values from the mean was less than +10%, except for spots with mobilities less than about 0.10. This methionine is not present in the cell-free product. § Trypsin does not cleave the bond between residues 57 and 58 in fl-MSH; hence this peptide could not be used as a marker. I This peptide was not clearly identified in the cell-free product due to incomplete tryptic cleavages in this region of the molecule.

peptides of the polysome runoff proteins will contain label of from the polysome runoff product relative to that in the pep- differing specific activity depending upon their positions in the tides derived from the de novo product should reveal the po- proteins. Hence, the amount of label in the peptides derived sitions of these peptides in the cell-free product. This method of locating peptides has been used successfully by Dintzis for rabbit hemoglobin (18). The presence of only one methionine and one tryptophan peptide in either mouse ACTH or ,B-LPH makes it possible to clearly interpret the polysome runoff experiment when either of these labels is used (1, 16, 17; unpublished work of J. L. E Q Roberts and E. Herbert). The electrophoretic profile of the C., [s5S]methionine-labeled tryptic peptides of the polysome runoff fn product (Fig. 4 upper), shows that the 3(61-69) LPH tryptic peptide labels more heavily than the a(1-8) ACTH tryptic peptide relative to the corresponding tryptic peptides derived from the mRNA-directed product. The ratio of cpm in polysome runoff peptides relative to cpm in de novo synthesized peptides (RNA-directed peptides) is 0.5 and 2.0 for a(1-8) ACTH and 13(61-69) LPH, respectively (legend of Fig. 4). The conclusion is the same for the [3H]tryptophan tryptic peptides of cell-free and 4 E ACTH fl-endorphin (Fig. lower). The ratio I of label in the peptides in this case is 0.9 for a(9-15) ACTH and 2.3 for 13(52-57) LPH. The other tryptophan-labeled peptides (Fig. 4 lower) have lower ratios ranging from 0.65 to 0.25. The same kind of studies have been done with isoleucine-, valine-, and phenylalanine-labeled peptides with similar results. These findings suggest that fl-LPH is located carboxy terminal to ACTH in the cell-free product and that ACTH is located carboxy terminal to the unidentified peptides in Fig. 4. Mobility, RLYS FIG. 4. Profiles of methionine- and tryptophan-labeled peptides DISCUSSION from the cell-free product made de novo and in polysome chain Two independent approaches have been used to demonstrate completion experiments. Cell-free ACTH was prepared as described in Materials and Methods and in Fig. 1. The RNA-directed methio- that the Mr 28,500 cell-free product contains the sequence of nine-labeled peptides, a(1-8) ACTH andB(61-69) LPH (Upper), both a(1-39) ACTH and fl-LPH. First, the Mr 28,500 poly- contain 310 and 290 cpm, respectively. The corresponding runoff peptides that are immunoprecipitated with antisera specific peptides contain 150 and 580 cpm for ratios of 0.5 (150/310) and 2.0 for ,B-endorphin and a(1-39) ACTH contain the same methi- (580/290). Tryptophan-labeled RNA-directed peptides, a(9-15) onine-, lysine-, and tryptophan-labeled tryptic peptides (Fig. ACTH and #(52-57) LPH (Lower), contain 190 and 170 cpm, respectively, and the corresponding runoff peptides contain 170 and 2). Second, tryptic digests of the product contain a number of 390 cpm for ratios of approximately 0.9 (170/190) and 2.3 (390/170). labeled peptides that behave like marker peptides from the [35S]Methionine (Upper) and [3H]tryptophan (Lower) tryptic pep- conserved regions of fl-LPH (Table 3) and porcine a(1-39) tides ofcell-free product synthesized de novo (O-*) or in a polysome ACTH during electrophoresis and chromatography. The amino chain completion experiment (O...O). acid composition data in Table 3 of this paper and table 1 of ref. Downloaded by guest on September 24, 2021 5304 Biochemistry: Roberts and Herbert Proc. Natl. Acad. Sci. USA 74 (1977) ACTH 3-LPH indicate that the ACTH protein also contains the sequence of NH2Ese 13-MSH ,, En COOH f3-LPH. Because no other class of proteins has been found to Pre sequence? ,B-MSH P-Endorphin contain ACTH or fl-LPH in the two kinds of translation ex- FIG. 5. Structure of the cell-free product. periments performed (Figs. 2 and 4), it seems highly likely that * Spacer peptide, length from 0 to less than 20 amino acids. the cell-free product is the precursor of all the forms of ACTH t COOH-terminal peptide of about five amino acids. and lipotropin found in AtT-20 cells (3, 9, 10). fl-MSH was a gift from the Ciba Corporation. Porcine fl-endorphin 1 confirm the identification of these peptides by showing that and the endorphin antiserum (RB-100) were a generous gift of Dr. they contain almost all of the amino acids present in the marker Roger Guillemin and his colleagues, Dr. Nicholas Ling and Ms. Vargo peptides. The simplest interpretation is that the cell-free of the Salk Institute, La Jolla, CA. This work was supported by National product contains the sequences of both a(1-39) ACTH and Institutes of Health Grant AM 16879. fl-LPH and that there is only one copy of each sequence present 1. Roberts, J. L. & Herbert, E. (1977) Proc. Natl. Acad. Sci. USA (Fig. 2 of this paper and figure 3 in ref. 1). 74,4826-4830. A definitive answer to the question of whether the complete 2. Eipper, B. A., Mains, R. E. & Guenzi, D. (1976) J. Biol. Chem. sequence of f3-LPH is contained in the precursor will have to 251,4121-4126. await sequencing work. However, recent studies suggest that 3. Mains, R. E. & Eipper, B; A. (1976) J. Biol. Chem. 251, 4115- a complete ,O-LPH-like molecule is cleaved from the Mr 31,000 4120. ACTH/f-LPH precursor during processing of this molecule 4. Phifer, R. F., Orth, D. 1. & Spicer, S. S. (1974) J. Clin. Endo- crinol. Metab. 39,684-695. in AtT-20 cells (ref. 9; and unpublished work of J. L. Roberts 5. Abe, K., Nicholson, W. E., Liddle, G. W. & Orth, D. N. (1967) and E. Herbert). In addition, we have observed several acidic J. Clin. Invest. 46, 1609-1620. tryptic peptides in the precursor that behave like peptides de- 6. Abe, K., Nicholson, W. E., Liddle, G. W., Orth, D. N. & Island, rived from the NH2-terminal region of f3-LPH (data not D. P. (1969) J. Cl. Invest. 48, 1580-1589. shown). 7. Gilkes, J. J. H., Bldomfield, G. A., Scott, A. P., Lowry, P. J., Rat- The results of the polysome runoff experiments (Fig. 4) cliffe, J. G., Landtn, J. & Rees, L. H. (1975) J. Clin. Endocrinol. suggest that jl3-LPH is located COOH-terminal to ACTH in the Metab. 40, 450-457. precursor. Because the precursor contains approximately 260 8. Li, C. H. & Chtng, D. (1976) Proc. Nati. Acad. Sci. USA 73, amino acids and ACTH and together account for only 1145-1148. f3-LPH 9. Mains, R. E., Eipper, B. A. & Ling, N. (1977) Proc. Natl. Acad. 130 amino acids, there are a number of possible locations for Sci. USA 74,3014-3018. these two hormones in the precursor. Several pieces of evidence 10. Giagnoni, G., Sabol, S. L. & Nirenberg, M. (1977) Proc. Natl. suggest that ACTH is located somewhere near the middle of Acad. Sci. USA 74,2259-2263. the precursor molecule and that fl-LPH is close to ACTH and 11. Martial, J. A., Baxter, J. D., Goodman, H. M. & Seeburg, P. H. near the COOH-terminal region of this molecule. First, all of (1977) Proc. Natl. Acad. Sci. USA 74, 1816-1820. the non a(1-39) ACTH and non fl-LPH peptides appear from 12. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, the pattern of label generated in the polysome runoff experi- 247-256. ments to be located NH2-terminal to ACTH (4 of the 8 tryptic 13. Pestka, S. (1971) Annu. Rev. Microbiol. 25,487-562. peptides in Fig. 4). The gradient of label in the runoff peptides 14. Herbert, E., Allen, R. A. & Paquette, T. L. (1977) Endocrinology, in press. also suggests that only a portion of one tryptic peptide (5 or 6 15. Guillemin, R., Ling, N. & Vargo, T. (1977) Biochem. Biophys. amino acids long) is located COOH-terminal to R3-LPH (data Res. Commun. 77,361-366. not shown). These observations are summarized in Fig. 5. 16. Mendez, E. & Lai, C. Y. (1976) Anal. Biochem. 65,281-292. Results in the first paper in this series (1) show that RNA and 17. Dayhoff, M. 0. (1972) in Atlas of Protein Sequence and Struc- polysomes from AtT-20 cells direct the synthesis of only one ture (National Biomedical Research Foundation, Washington, ACTH protein in the reticulocyte cell-free system with an ap- DC), Vol. 5, pp. D-194-D-197. parent molecular weight of 28,500. The results in this paper 18. Dintzis, H. M. (1961) Proc. Natl. Acad. Sci. USA 47,247-261. Downloaded by guest on September 24, 2021