Biosynthesis of Adrenocorticotropic Hormone in Mouse Pituitary Tumor Cells *

A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor ccl! line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell extracts resolved four forms of ACTH with apparent molecular weights of 4,500, 13,000, 23,000, and 31,000. These four forms of ACTH can be detected by radioimmunoassay of cell extracts or by immunoprecipitation of cell extracts following incubation of cultures in [3H]tryptophan, [‘j-fllysine, or [3H]tyrosine. The double antibody immunoprecipitation scheme developed is specific, quantitative, and reproducible. ACTH biosynthesis was examined in both steady and pulse-labeling experiments using [8H]tyrosine or [3H]lysine. The results of these experiments are consistent with the proposal that M, = 31,000 ACTH is the biosynthetic precursor for all three smaller forms of ACTH and that M, = 23,000 ACTH is a biosynthetic intermediate. Both M, = 13,000 ACTH and M, = 4,500 ACTH are derived from the intracellular processing of M, = 3 1,000 ACTH.

From the Departments of Biology and Chemistry, University of Oregon, Eugene, Oregon 97403 A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor ccl! line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell extracts resolved four forms of ACTH with apparent molecular weights of 4,500, 13,000, 23,000, and 31,000. These four forms of ACTH can be detected by radioimmunoassay of cell extracts or by immunoprecipitation of cell extracts following incubation of cultures in [3H]tryptophan, ['j-fllysine, or [3H]tyrosine. The double antibody immunoprecipitation scheme developed is specific, quantitative, and reproducible. ACTH biosynthesis was examined in both steady and pulse-labeling experiments using [8H]tyrosine or [3H]lysine. The results of these experiments are consistent with the proposal that M, = 31,000 ACTH is the biosynthetic precursor for all three smaller forms of ACTH and that M, = 23,000 ACTH is a biosynthetic intermediate. Both M, = 13,000 ACTH and M, = 4,500 ACTH are derived from the intracellular processing of M, = 3 1,000 ACTH.
High molecular weight forms of adrenocorticotropic hormone have been observed in pituitary extracts and pituitary tumor extracts from several different species (l-9). The ACTHIsecreting mouse pituitary tumor cell line, AtT-20/D-16v, contains several high molecular weight forms of ACTH (1) and provides a convenient experimental system in which to investigate the function of these forms in the biosynthesis of ACTH. Tumor cells were incubated with 'H-amino-acids and a double antibody immunoprecipitation scheme was used to separate SH-labeled ACTH from other Wlabeled cell proteins.

Resolution of Forms of ACTH
on SDS Gels-Earlier studies using gel filtration in guanidine HCl resolved the ACTH activity in normal mouse pituitaries and in the AtT-20/Dl6v cells into three size classes: M, = 4,000 to 5,500 ACTH, M, = 6,500 to 9,000 ACTH, and M, = 20,000 to 30,000 ACTH (1, 2). The use of SDS-polyacrylamide gel electrophoresis (18) has made it possible to achieve better resolution of the different forms of ACTH (Fig. 1). Reference to peaks of ACTH immunoactivity will be based on their apparent molecular weight in this gel system. ACTH activity in the M, = 20,000 to 30,000 size class was resolved into two distinct forms: M, = 23,000 ACTH and 31,000 ACTH. Cell extracts and tissue culture medium both contained M, = 23,000 ACTH and 31,000 ACTH; there was relatively more M, = 23,000 ACTH in the medium than in the cell extracts. The peak of M, = 13,000 ACTH seen in both cell extracts and culture medium corresponds to the M, = 6,500 to 9,000 ACTH previously described; this substantial difference in apparent molecular weight is considered further in the accompanying paper (23). As was seen using gel filtration in guanidine HCl, the predominant form of ACTH in cell extracts comigrated with Y-cr,(l-39) (M, = 4,500 ACTH), but there was little if any of this form of ACTH in the culture medium. It has not been determined whether the differences between the ACTH pattern in cell extracts and in culture medium are due to selective secretion of variousforms, selective proteolysis, or interconversion after secretion. In studying the kinetics of labeling of the four forms of ACTH, it is necessary to take into account ACTH secreted into the culture medium. For example, after the 6 hour incubation in [*H]lysine (Fig. 3C), the medium contained approximately the same amount of BH-labeled immunoprecipitable material as the cell extract (data not shown). However, at the short times used for pulse-labeling experiments (see below), the amount of labeled ACTH secreted is not significant; after 30 min there was less than 1% as much $H-labeled immunoprecipitable material in the medium as in the cells, and after 2 hours that ratio was about 10%.
Pulse-labeling Experiments-In order to expose the cells to a short pulse of 8H-labeled amino acid with minimal disturbance to the system, the concentration of amino acids present in normal growth medium was used throughout the pulse and chase periods. Fig. 4 shows the results of exposing the .'.tT-20 cells to a 20-min pulse of [BH]tyrosine and then analyzing the cultures after varying periods in nonradioactive medium. Immunoprecipitation of a sample exposed to [sH]tyrosine for 20 min showed that label had been incorporated only into M, = 31,000 ACTH (Fig. 4A). After a 20-min chase incubation in unlabeled tyrosine (Fig. 4B)  Approximately 5 ng of RIA-ACTH activity were applied to each gel. For short incubation times, immunoprecipitable counts represented 0.6% of the trichloroacetic acid-insoluble radioactivity. 0, specific immunoprecipitate; performed using Solution F; 0, a(l-24) competed immunoprecipitate; performed using Solution G. label appeared in M, = 13,000 ACTH and 4,500 ACTH more slowly than in the two higher molecular weight forms of ACTH; M, = 13,000 ACTH did not label and chase more rapidly than M, = 4,500 ACTH. The results obtained with [SH]lysine and [8H]tyrosine were identical. DISCUSSION In order to examine the kinetics of synthesis of ACTH, it is necessary to purify all four forms of ACTH away from all other cell products in high yield. The immunoprecipitation procedure described here was shown to be reproducible, specific (JH-labeled material was displaced by of( l-24)) and quantitative. In addition, pituitaries are known to contain enzymes that can destroy ACTH activity (9, 25); therefore the protease inhibitors phenylmethylsulfonyl fluoride and iodoacetamide were used in these studies to prevent degradation or interconversion during the extraction and immunoprecipitation procedures. Pulse-chase experiments were performed without protein synthesis inhibitors; the rapid equilibration of cellular amino acid pools with the tissue culture medium indicated that a pulse of label could be introduced into the biosynthetic pathway simply by replacing the culture medium. The fact that the amount of label in M, = 31,000 ACTH decreased steadily throughout the incubation in unlabeled amino acid indicates that the chase procedure was effective; in the continued presence of radioactive amino acids, M, = 31,000 ACTH would have continued to label linearly for at least 40 min (Fig. 3). trichloroacetic acid-insoluble counts (incorporated during the brief incubation in [SH]tyrosine) disappeared during incubation in unlabeled growth medium with a half-life of 3 to 4 hours. 0, specific immunoprecipitate; performed using Solution F; 0, a(l-24) competed immunoprecipitate; performed using Solution G.
Howe\ver, extracts of mouse anterior pituitary also contain higtr molecular weight forms of ACTH (2), and it is anilcipated that a similar biosynt,hetic pathway will function in the mouse pituitary. It is not yet known whether any of the high molecular weight forms of ACTH are normally secreted by the pituitary and thus might have a functional role in the periphery in addition to their presumptive role as intermediates in the biosynthesis of M, = 4,500 ACTH. CHASE PERIOD, minutes into M, = 31,000 ACTH during a 20-min pulse-labeling period appears in M, = 13,000 ACTH plus 4,500 ACTH after a chase period of 45 to 60 min, so the conversion process appears to be quite efficient. The AtT-20 cell line will be useful for studying other aspects of ACTH biosynthesis, such as the structural relationships of the forms of ACTH, the polypeptide fragments created during conversion of 31,000 ACTH to the smaller forms of ACTH, and the enzymes involved in the conversion process.
synthesis of M, = 31,000 ACTH. The high molecular weight forms of ACTH are not simply aggregates of M, = 4,500 ACTH since they maintain their integrity after reduction and fractionation in the presence of SDS or guanidine HCl (1). The results of pulse-chase and steady labeling experiments (Figs. 3 to 5) are consistent with the suggestion that M, = 31,000 ACTH is the biosynthetic precursor to all three smaller forms of ACTH. The kinetics of labeling of M, = 23,000 ACTH suggest that it is a biosynthetic intermediate in this pathway. The quantitative question of whether all M, = 31,000 ACTH molecules are processed through the M, = 23,000 ACTH intermediate or whether some M, = 31,000 ACTH can bypass the M, = 23,000 ACTH intermediate cannot be answered yet. The data presented indicate that M, = 13,000 ACTH and 4,500 ACTH are derived from a biosynthetic precursor (M, = 31,000 ACTH and possibly 23,000 ACTH), but it is not yet clear whether M, = 13,000 ACTH can or must serve as a precursor to M, = 4,500 ACTH. The present data indicate that the processing of M, = 31,000 ACTH to 13,000 ACTH and 4,500 ACTH may be similar to the processing of proinsulin to insulin (26) and of proparathyroid hormone to parathyroid hormone (27). The products of the in vitro translation of ACTH mRNA will have to be studied in order to determine whether ACTH biosynthesis involves a short-lived precursor analogous to preimmunoglobulin (28), preproparathyroid hormone (29, 30), and pregrowth hormone (31).
Hormone-secreting tumors have often been found to differ from normal tissue in the details of intracellular processing.