Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.

into labeled proteins similar to /l-endorphin and cY-melanocyte-stimulating hormone.
In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium. Previous work defined the biosynthetic pathways for adrenocorticotropic hormone (ACTH, corticotropin)' and P-endor-* This work was supported by National Institutes of Health Grants AM-19859 and AM-18929. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC.
Section 1734 solely to indicate this fact. ' The abbreviations used are: ACTH, adrenocorticotropic hormone phin in the mouse pituitary tumor cell line, AtT-20/D-16v. A diagram of the structure of the common precursor molecule and the proteolytic cleavages involved in creating ACTH and P-endorphin is shown in Fig. 1 (l-6). Cell-free protein synthesis has confirmed the existence of a common precursor to ACTH and ,B-endorphin in the AtT-20 tumor and in rat and bovine pituitaries and has shown that the ACTH-and pendorphin-containing primary gene product is very similar in the tumor cells and in the anterior and intermediate and corticotropin-like intermediate lobe peptide (CLIP; ACTH(18-39)), which do not occur in substantial amounts in the anterior lobe; the intermediate lobe contains primarily P-endorphin and relatively little P-lipotropin (PLPH), while the anterior lobe contains more /3LPH than /3-endorphin (18-26). Since the two lobes of the pituitary synthesize very similar primary gene products but contain very different collections of final products derived from the larger molecule, it seems likely that tissue-specific differences in proteolytic enzymes and other biosynthetic processing enzymes are crucial in generating the differences observed between the lobes.
Quantitative analyses of pulse-chase experiments with the AtT-20/D-16v tumor cells have shown that each molecule of pro-ACTH/endorphin that undergoes proteolytic processing generates a smaller ACTH-related molecule and a smaller /3endorphin-related molecule (5). As expected, synthesis and release of ACTH-related and P-endorphin-related proteins are closely coupled in the basal state, during glucocorticoid feedback inhibition, and during stimulation by hypothalamic corticotropin-releasing factor or by cyclic nucleotides (18,19,(27)(28)(29). ACTH and P-endorphin release from normal anterior pituitary tissue is regulated by the same factors that affect hormone release from the tumor cells (18,(27)(28)(29)(30)(31)(32) The structures and biosynthetic interrelationships of the molecules shown have been summarized from Refs. 1 to 6. Pro-ACTH/endorphin and ACTH biosynthetic intermediate occur in forms that differ by the addition or lack of addition of an oligosaccharide chain within their ACTH(l-39)-like sequence; the different forms can be separated by SDS-gel electrophoresis and by gel filtration. The average apparent molecular weights from SDS-polyacrylamide gel electrophoresis ("K" nomenclature) and from gel fitration in 6 M guanidine HCl ("Mr =" nomenclature) are as follows: pro-ACTH/endorphin, 30K or M, = 26,500 (average of both forms); ACTH biosynthetic intermediate, 22K or M, = 16,500 (average of both forms); PLPH,11.7K or M,= 8,200;16K fragment,16K or M,= 11,200;, 13K or M, = 6,700, nonglycosylated ACTH( l-39), 4.5K or M, = 4,500; rLPH, 8K or M, = 4,600; P-endorphin, 3.5K or M, = 3,500. otMSH is Nacetyl-ACTH(l-13)NH2 and CLIP is ACTH(18-39); (uMSH and CLIP are not major products of the AtT-20/D-16v tumor cells or of anterior pituitary, but are major products in intermediate pituitary. studies on a cell suspension to be representative of the behavior of the tissue in situ, the yield of viable cells must be high enough so that it is clear that an abnormal subpopulation of cells is not being studied. In addition, the viability of the cells must not decrease markedly during the period of study. in 30 h and were thus stable enough for study. These results do not, however, yield any information about ' "Methods", some "Results" (including Table I &cl Figs. 2,5,7,11,and 13), and all of the references are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journal of Riological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Reauest Document No. 79111-335. cite author(s). and include a check or money order for $2.25 per set of photocopies. the molecular forms of the immunoactive proteins or about their rates of synthesis. Fig. 3 shows an analysis of two aliquots of an intermediate pituitary cell suspension after incubation in medium containing [3H]phenylalanine for 6 h; one sample (short term cells) was incubated in radioactive medium immediately after dissociation, and one sample (long term cells) was preincubated for 24 h before labeling. The molecular forms of NH2-terminal ACTH-related, 16K fragment-related, and NHs-terminal P-endorphin-related material synthesized by short term cells and long term cells were similar. More detailed analyses of the peptide products would be required to determine whether subtle changes in processing occurred during the time in culture (such as alterations in the degree of N-acetylation of the LuMSH-like material in Fig. 3A ). The major products one would expect to be produced from a pro-ACTH/endorphin-like molecule, based on the mouse pituitary tumor cell work ( molecule appeared to undergo a greater amount of proteolytic processing in the intermediate lobe cells than it did in the AtT-20 cells. This further proteolytic processing of 16K fragment cannot yet be interpreted, since the specificity of the 16K fragment antibody is not known. In various incubations of intermediate pituitary cells with labeled precursors, the ratio of total immunoprecipitable radioactivity (sum of all pro-ACTH/endorphin antibodies) to total acid-precipitable radioactivity (12.5% trichloroacetic acid) varied from 5 to 20% depending on the labeled amino acid used. In a number of studies by other workers, the amount of /?-endorphin, /3MSH, CLIP, or aMSH in the intermediate lobe was found to be 2 to 5 pmol of peptide/pg of protein (20, 22-24, 36, 52-60). Using the fact (see below) that /?-endorphin and aMSH are produced from a molecule similar in size to tumor cell pro-ACTH/endorphin (with a molecular weight during gel filtration in 6 M guanidine HCl of about 26,500), it can be calculated that roughly 10% of the protein in the intermediate lobe should be immunoprecipitable with the appropriate pro-ACTH/endorphin antibodies:] When an aliquot of short term cells identical with the cells used in Fig. 3  of 31,000,13,000, and 3,500; these peaks correspond to mouse tumor cell pro-ACTH/endorphin, PLPH, and P-endorphin, respectively. The middle ACTH antibody was added to the supernatant from this first immunoprecipitation and detected only three pools of ACTH with apparent molecular weights of 22,000,14,000, and 4,500. When a second aliquot of this cell extract was analyzed by using the middle ACTH antibody before the P-endorphin antibody, the 31K peak was found in the middle ACTH immunoprecipitate and was absent from the P-endorphin immunoprecipitate; the results of this crossed immunoprecipitation experiment show that one 31K molecule contains antigenic determinants for both ACTH and P-endorphin.
When the 16K fragment antibody was added to the supernatant remaining after immunoprecipitation with the NH&erminal P-endorphin and middle ACTH antibodies, it detected a broad peak of labeled material with an apparent molecular weight of 16,000; thus antibody to mouse tumor cell 16K fragment detected a similar cross-reactive protein in rat pituitary. When a separate aliquot of the same cell extract was analyzed by using the 16K fragment antibody as the initial antibody, the 3lK and 22K peaks appeared in the 16K fragment immunoprecipitate.
These results are consistent with a model for the structure of the rat 31K molecule that is similar to the model for the structure of mouse tumor cell pro-ACTH/endorphin shown in Fig. 1. Finally, when the NHzterminal ACTH antibody was added to the supernatant remaining after immunoprecipitation with the three antibodies described above, the NH&erminal ACTH antibody immunoprecipitated only a single peak of material. On SDS-polyacrylamide gels this peak migrated at the position of lz51labeled aMSH; when analyzed by gel filtration in 6 M guanidine HCl, this material eluted near the position of (YMSH and thus appeared to contain 13 to 14 amino acids. When analyzed in a similar way, AtT-20/D-16v tumor cells did not produce significant amounts of aMSH-like material. When the NHzterminal ACTH antibody was used as the initial antibody, a pattern similar to that shown in Fig. 3A  and a smaller cross-reactive peak at 5K; peaks at the expected positions for CLIP and glycosylated CLIP were seen using the middle ACTH antibody after the NHz-terminal ACTH antibody (5K and 13K, respectively).
For the incubations shown in Figs. 4 and 5 it is possible to estimate how much of the ACTH-and ,8-endorphin-related protein in the cells became labeled during the incubation period. The P-endorphin content of the sample analyzed in Fig. 4 was estimated from Table I produced glycoprotein forms of ACTH and 16K fragment, cells were incubated with [3H]glucosamine and the proteins synthesized were analyzed by the sequential immunoprecipitation technique (Fig. 6). The three high molecular weight forms of ACTH and 16K fragment were found to be glycoproteins. As expected, no labeled glucosamine was incorporated into 4.5K ACTH, the cuMSH-like molecule (2K), the ,8LPHlike molecule, or the ,&endorphin-like molecule. Thus the pattern of glycosylation of the rat intermediate pituitary 30K molecule appeared to be qualitatively similar to the pattern of glycosylation of mouse tumor cell pro-ACTH/endorphin (Fig. 1)

Thus the initial intracellular product in rat intermediate pituitary cells contained antigenic determinants
for several of the peptide regions predicted based on the mouse tumor cell pro-ACTH/endorphin model (Fig. 1). Final Products from the Common Precursor-The initial intracellular product observed above could serve as a precursor to a wide variety of smaller protein products; before analyzing the details of the intermediate steps in the biosynthetic pathways, the end points of the processing pathway were determined.
In an experiment similar to the one in Fig.  8 for 15 mm; one ahquot of cells was extracted immediately (pulse) and an equal aliquot was incubated for 6 h in complete nonradioactive culture medium before extraction (chase). Samples were immunoprecipitated with various antibodies and analyzed on SDS-polyacrylamide gels (Fig. 9). After the 15min pulse, the endorphin antiserum immunoprecipitated only the expected peak of 30K material. Following the 6-h chase incubation, antibodies to ACTH, P-endorphin, and 16K fragment did not detect any radioactivity remaining at the 30K position; molecules similar to P-endorphin, aMSH, and 16K fragment were detected as final intracellular products. There are 3 methionine residues in mouse tumor cell pro-ACTH/endorphin: one at position 5 of P-endorphin, one at position 4 of ACTH, and one in the middle region of 16K fragment (6); based on analyses of tryptic and chymotryptic peptides, the methionine content of the rat pituitary 30K molecule appears to be similar.5 After the 6-h chase period ( Fig. 9B), the P-endorphin immunoprecipitate contained a single ["%]methionine-labeled peak with an apparent molecular weight of 3.5K; this 3.5K peak contained 31% of the radioactivity present in the immunoprecipitable 30K pool after the 15-min pulse. with a protein as large as /3LPH (12K in this gel system) was found. Based on gel filtration in 6 M guanidine HCl, less than 10% of the labeled 3.5K endorphinrelated material was as small as cu-endorphin.
After a 6-h continuous labeling period in [35S] The NH&erminal ACTH antibody immunoprecipitated a single peak of ["'Slmethionine-labeled material that co-migrated with 12?-labeled aMSH (Fig. 9B); this aMSH-like molecule contained 38% of the radioactivity initially present in the immunoprecipitable 30K pool. The aMSH-like molecule was 13 to 14 amino acids in length, as judged by gel filtration in 6 M guanidine HCl. Paper electrophoretic analyses (pH 6.35 and pH 3.5) of the tryptic peptides of the aMSH-like molecule labeled with r3H]phenylalanine or with [35S]methionine were consistent with the suggestion that most (but not ah) of the molecules had an acetylated NH2 terminus (aMSH is N-acetyl-ACTH( l-13)NH2); substantial amounts of aMSHlike molecules lacking N-acetylation have been purified from camel and dogfish pituitaries (59,67). No peaks of radioactivity were detected at the positions of ACTH(l-39) (4.5K) or glycosylated ACTH( l-39) (13K); these labeled molecules would appear in the NH2-terminal ACTH immunoprecipitate if they were present in the extract (e.g. Figs. 3 and 5). When a separate aliquot of the 6-h chase sample was analyzed with the middle ACTH antibody, the immunoprecipitate contained only about 1% of the radioactivity initially present in the immunoprecipitable 30K pool (Fig. 9A). Thus aMSH and presumably CLIP (which would not be labeled by [3"S]methionine) were the final ACTH-related products synthesized by rat intermediate pituitary cell suspensions; in contrast, the major products produced by mouse pituitary tumor cells contained an intact ACTH( l-39)-like sequence.
The 16K fragment antibody also immunoprecipitated some [35S]methionine-containing material from the 6-h chase extract (Fig. 9B); the peaks, with apparent molecular weights of 16K and 4K, contained only 18% of the radioactivity immunoprecipitable after the 15-min pulse. The relatively low yield of labeled 16K fragment-related material present after the 6h chase may have been due to conversion of 16K fragment into smaller [?S]methionine-containing peptides that were not recognized by the antibody. Until the location of the methionine residue in 16K fragment and the specificity of the antiserum are known, this question will remain unresolved.
About 87% of the radioactivity present as immunoprecipitable 30K material after a 15-min pulse was found in the cells as immunoprecipitable smaller proteins after a 6 h chase; only about 4% of the initial immunoprecipitable radioactivity appeared in the culture medium during the 6-h chase. Thus nearly all of the newly synthesized precursor was converted into a distinct set of smaller molecules.
Samples of the whole cell extract (without prior immunoprecipitation or trichloroacetic acid precipitation) were also analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 9); as expected, a heterogeneous collection of newly synthesized proteins was observed. After the 15-min pulse, 12% of the [?S]methionine-labeled material on the gel was immunoprecipitated by the ,&endorphin antibody. Although a peak at 30K could be discerned in the analysis of the whole cell extract, only half of the 30K material was immunoprecipitated by any of the pro-ACTH/endorphin antibodies. After the 6-h chase, there was still labeled material in the whole cell extract at the 31K position, but none of the pro-ACTH/endorphin antibodies detected any immunoprecipitable labeled molecules of that size. After the 6-h chase, the whole cell extract contained a peak at 3.5K; the aMSH-like molecule, P-endorphin-like molecule, and some of the 16K fragment-related material found after the 6-h chase incubation all contributed to formation of this peak of labeled low molecular weight material observed in the whole cell extract (Fig. 9B).
Further Analysis of the Biosynthetic Pathway of ACTH and &SH-The results in Figs. 7 to 9 showed that a single 30K molecule was the common precursor for several smaller proteins in the intermediate lobe of the rat pituitary. In order to understand the steps involved in converting the approximately 30,000-dalton precursor molecule into 1500-to 4500dalton products, additional experiments were performed. Several aliquots of rat intermediate pituitary cells were incubated in medium containing [3H]phenylalanine for progressively increasing periods of time and two different ACTH antibodies were used to determine the kinetics of appearance of labeled ACTH-related proteins (Fig. 10). The peak of immunoprecipitable material at 31K built up in time and reached a plateau level; after labeled 31K material appeared in the cells, radioactivity appeared at the 22K position and built up to a plateau level. The 31K molecule corresponds to mouse pituitary tumor cell pro-ACTH/endorphin and the 22K peak corresponds to ACTH biosynthetic intermediate (Fig. 1). When the middle ACTH antibody was used, peaks of radioactivity at 14K and 4.5K continued to accumulate for the entire 12-h incubation. The NHz-terminal ACTH antibody precipitated an aMSHlike molecule from the supernatant left after precipitation with the middle ACTH antibody.
The intact 14K and 4.5K molecules had masses of 4600 and 2800 daltons, respectively, during gel filtration in 6 M guanidine HCl and thus were significantly smaller than the glycosylated ACTH (l-39) and ACTH( l-39) found in the mouse pituitary tumor cells. Rat ACTH (l-39) would be expected to contain phenylalanine residues at positions 7, 35, and 39 (4, 24, 68).  Fig. 4; cells were harvested at the indicated times and extracts (0.0017 of a lobe) were immunoprecipitated with middle ACTH antibody and analyzed using borate-acetate-buffered SDS-polyacrylamide gel electrophoresis (10% acrylamide).
Culture medium from the cells (corresponding to 0.034 of a lobe) was also immunoprecipitated and analyzed; for the samples of culture medium, the radioactivity above background (20 cpm) was divided by 20 and plotted to correspond to the amount of cell extract analyzed.
The olMSH-like molecules in the cell extracts were isolated by using the NHz-terminal ACTH antibody sequentially after the middle ACTH antibody. and 4.5K material present after 12 h of steady labeling were analyzed by paper electrophoresis at pH 6.35 and pH 3.5 and by gel filtration in 6 M guanidine HCl. For both the 14K and 4.5K molecules, most (~35%) of the radioactivity was present in a large, anionic tryptic peptide similar to glycosylated and nonglycosylated ACTH(22-39), respectively (4, 6); the tryptic digests contained little radioactivity that could be attributed to the ACTH(l-8) tryptic peptide. Thus both of these peaks contained segments similar to the COOH-terminal half of ACTH (l-39) and both lacked segments similar to the NH2terminal end of ACTH( l-39). The ["Hlphenylalanine-containing tryptic peptides derived from the 14K and 4.5K peaks had masses of 3900 and 2000 daltons, respectively. Thus the 14K and 4.5K molecules in Fig. 10 (and similarly in Fig. 5, open boxes) are similar to glycosylated and nonglycosylated CLIP (ACTH( 18-39)). Since ACTH( 17-39) has also been isolated from human pituitary extracts (25), the exact structure of the 14K and 4.5K ACTH-related peaks will require more detailed peptide analyses.
During the 12-h incubation a small percentage of the newly synthesized ACTH-related molecules appeared in the culture medium (Fig. 10); basal release of hormone into the culture medium is examined in more detail below.
Further Analysis of the Biosynthetic Pathway of /?-Endorphin-In Fig. 11, the NH&erminal ,&endorphin antibody was used to analyze the same steady labeling experiment analyzed with ACTH antibodies in Fig. 10. A peak of immunoprecipitable material at 30 to 31K was again found to build up and reach a steady level. After labeled 31K material appeared in the cells, a PLPH-like protein (13K) became labeled and built up to a plateau. Radioactivity continued to collect in a 3.5K peak (which co-migrated with '"'I-labeled &-endorphin) for the entire incubation period. The 3.5K material eluted at the position of ""I-labeled ,L?,-endorphin during gel filtration in 6 M guanidine HCl.
In order to determine whether the PLPH-like molecule served as a biosynthetic intermediate on the pathway from the 31K molecule to 3.5K endorphin, pulse-chase experiments were carried out (Fig. 12) rat /3LPH-like and P-endorphin-like molecules has shown that both of these molecules contain only 1 methionine residue.5 After the pulse, the only labeled immunoprecipitable molecule present was the common precursor (29K). After a l-h chase in nonradioactive medium a substantial amount of labeled precursor was converted into a PLPH-like molecule (12.5K), but no labeled /3-endorphin (3.5K) had appeared. After a 2-h incubation in unlabeled medium, the 3.5K endorphin became labeled. Finally, after a 6-h chase period, 3.5K endorphin was the only labeled P-endorphinrelated molecule remaining in the cells (the same result as in Fig. 9). This result shows that a PLPH-like molecule is an obligatory biosynthetic intermediate in the production of a P-endorphin-like molecule from the 30K common precursor in rat intermediate pituitary cells. In the pulse-chase experiment shown in Fig. 12, 34% of the radioactivity present in the 30K common precursor after a 15-min pulse was detected in the 3.5K endorphin product after a 6-h chase. In three separate pulse-chase experiments (one with ["Hlphenylalanine and two with [35S]methionine), the half-life of the 30K common precursor during the chase periods was about 30 min in the rat intermediate pituitary cells. If the processing of the rat pituitary PLPH-like molecule into a P-endorphin-like molecule follows the scheme outlined in Fig. 1, a yLPH-like molecule should be created as ,&LPH is cleaved to /3-endorphin; a direct demonstration of this expected result is shown in Fig. 13. The yLPH-like molecule (6 to 6.5K) was further processed into smaller immunoprecipi- 14. Analysis of radiolabeled proteins appearing in culture medium during a pulse-chase experiment.
The [35S]methionine-containing molecules appearing in the culture medium during the pulse-chase experiment described in Fig. 9 were examined by sequential immunoprecipitation with NHz-terminal ACTH antibody, 16K fragment antibody, and NHn-terminal P-endorphin antibody. Medium corresponding to 0.25 of a lobe was used here; immunoprecipitates were analyzed on 11.25% acrylamide borate-acetate-buffered SDS-gels with dansylated cytochrome c as internal marker ($). The total immunoprecipitable radioactivity appearing in the medium after 6 h was 1042 cpm or 4% of the immunoprecipitable radioactivity in the cell extracts after the 15-min pulse. neural innervation.
Using the rat intermediate pituitary cell suspensions, the basal rate of appearance and molecular form of radiolabeled hormones in the medium were determined. The [35S]methionine-labeled ACTH-, ,&endorphin-, and 16K fragment-related proteins which appeared in the culture medium during a pulse-chase incubation are shown in Fig. 14. Under basal conditions, only a small percentage of the intracellular labeled hormone stores were released into the medium (approximately 4%/6 h); the sample of medium analyzed in Fig. 14 corresponded to 10 times the amount of cell extract analyzed in Fig. 9. As expected for healthy cells which were not simply leaking their hormone content into the medium nonspecifically, no secretion of newly synthesized labeled hormone occurred during the first 15 min of the chase period. After 45 min, a substantial amount of the 29 to 31K precursor was secreted and only small amounts of other ACTH-, ,L?endorphin-, or 16K fragment-related molecules appeared in the medium. After 6 h in the nonradioactive chase medium, a little more of the precursor appeared in the medium along with substantial amounts of each of the smaller ACTH-, aMSH-, ,B-endorphin-, and 16K fragment-related molecules. In a basal state, the isolated rat intermediate pituitary cells secreted the various smaller peptide products derived from the common precursor in a coordinated manner; secretion of ACTH-related products (aMSH, CLIP, ACTH), PLPH-related products (PLPH, ,&endorphin, yLPH), and 16K fragment-related products occurred at roughly equimolar rates.

Intermediate pituitary cell suspensions and immunoprecipitation techniques
were used to study the biosynthesis of ACTH-and P-endorphin-related proteins in the intermediate pituitary. The initial intracellular product was a glycoprotein (apparent molecular weight during SDS-polyacrylamide gel electrophoresis of about 30K) that carried antigenic determinants for all of the major segments of the AtT-20 mouse pituitary tumor cell pro-ACTH/endorphin molecule: /3-endorphin, yLPH, ACTH(17-24), ACTH(l-16), and 16K fragment. Kinetic studies demonstrated that this 30K molecule was processed intracellularly into a discrete collection of products including molecules similar to &endorphin, mouse tumor cell yLPH, aMSH, CLIP, and 16K fragment. As was found previously with the mouse pituitary tumor cells, the higher molecular weight forms of ACTH and 16K fragment were glycopro-teins; a molecule with the properties expected of glycosylated CLIP was also identified.
At the present time we are hesitant to assign a name to the 30K common precursor in the intermediate pituitary.
In the mouse anterior pituitary tumor cells (AtT-20/D-16v), a similar 30K molecule has been called pro-ACTH/endorphin by analogy to proinsulin and proparathyroid hormone; this molecule serves as a precursor to ACTH, which has a clear role in physiology, and P-endorphin, which may also have effects as a circulating hormone ( peptides; a complete culture medium was utilized and the level of labeling achieved for aMSH and CLIP using [14C]proline for 6 h was roughly 2% of the theoretical maximum (68). The studies reported here, using dispersed cells, were also carried out in complete culture medium, and the level of labeling of the various hormonal products reached at least 10% of the theoretical maximum after 12 to 16 h of incubation. Much lower levels of labeling (less than 0.005% of the theoretical maximum) were obtained in other studies when intermediate pituitary slices or cells were incubated in a simple saline solution containing glucose, albumin, and a single radioactive amino acid (43)(44)(45)(73)(74)(75). These quantitative differences may reflect the dramatic and rapid changes that occur in mRNA and tRNA pools, amino acid transport, protein synthesis and breakdown, and lysosomal activity in tissues deprived of adequate amino acid supplies (76-80).
The early steps in the intracellular processing of the 30K common precursor molecule in intermediate pituitary tissue are similar to the processing pathway defined in the AtT-20 mouse pituitary tumor cells (Fig. 1). However, intermediate pituitary cells carry out several important additional steps of processing. As predicted by Scott et al. (22), in the intermediate lobe cells the ACTH-like segment of the 30K precursor is cleaved to yield molecules similar to aMSH and CLIP. These biosynthetic results are consistent with the fact that the amount of intact, biologically active ACTH(l-39) found in intermediate pituitary extracts is only a few per cent of the amount of aMSH or CLIP found in this tissue (20,(22)(23)(24)36,56,60,68,81,82).
In the AtT-20 cells and in anterior pituitary tissue there is very little production of aMSH or CLIP and most of the ACTH(l-39) segment is kept intact.