further analysis of post-translational processing of p-endorphin in

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 256, No. 11, I sue of June 10, pp. 5689-5695, 1981

Further Analysis of Post-translational Processing of P-Endorphin in Rat Intermediate Pituitary*

(Received for publication, November 3, 1980, and in revised form, January 9, 1981)

Betty A. Eipper and Richard E. Mains From the Department of Physiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Rat intermediate pituitary cells maintained in culture synthesize the same forms of /I-endorphin observed in intermediate pituitary extracts. Biosynthetically labeled intermediate pituitary fl-endorphin-sized material was fractionated by ion exchange chromatography on sulfopropyl-Sephadex and the identities of the major peaks were determined by co-chromatography with synthetic marker peptides, gel filtration, and analysis of pronase, chymotrypsin, and trypsin digests. Peaks of a-N-acetyl-/I-endorphin(l-27), a-N-acetyl-&endorphin- (I-31), and P-endorphin(1-31) were identified and a fourth peak (eluting from the sulfopropyl-Sephadex column at 0.18 M NaCI) was tentatively identified as a- N-acetyl-#3-endorphin(1-26). Analysis of /3-endorphin synthesized in the presence of [36S]methionine and [3H]histidine confirmed the absence of Hisz7 in the material eluting at 0.18 M NaC1. Based on both steady labeling and pulse-chase incubations, /I-endorphin(1- 31) was the first form of labeled @endorphin-sized material to appear in cell extracts. This molecule was quickly N-acetylated on its NH2-terminal tyrosine residue and was then more slowly converted to a-N-acetyl- fi-endorphin(1-27) and then to cu-N-acetyl-P-endorin- (1-26).

Cells in both the anterior and intermediate lobes of the rat pituitary produce 6-endorphin-sized material by proteolytic cleavage of an approximately 30,000-dalton precursor molecule called pro-ACTH’/endorphin (1-3). As shown by Smyth and Zakarian (4), ion exchange chromatography can be used to resolve the /?-endorphin-sized material into several components. In the previous paper, we demonstrated that in rat anterior pituitary, the major form of P-endorphin-sized material was similar to synthetic ,Bc-endorphin(l-31) (5). In rat intermediate pituitary, materid identical with &endorphin(1-31) was found to be a minor component; the &endorphin-sized materid was resolved into a complex collection of forms. In both rat pituitary (6) and pig pituitary (7,8), Smyth and eo-workers identified the major forms of ,&endorphin- sized material as a-N-acetyl-/3-endorphin(l-27) and P-endorphin(1-27). In the course of studying the biosynthesis of p- endorphin in rat intermediate pituitary cell suspensions, we realized that P-endorphin(1-27) was not a major product in rat intermediate pituitary cells. In this study, we present data on the kinetics of biosynthesis of the multiple forms of rat

* This work was supported by National Institutes of Health Grants AM-I8929 and AM-19859. 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 U.S.C. Section 1734 solely to indicate this fact.

‘ The abbreviations used are: ACTH, adrenocorticotropin; SP- Sephadex, sulfopropyl-Sephadex; PLPH, P-lipotropin; &endorphin, camel P-endorphin.

intermediate pituitary @-endorphin and data on the identification of the major P-endorphin-related products.

METHODS’

RESULTS

Comparison of Immunoactive and Biosynthetically Labeled p- Endorphin

As demonstrated by Zakarian and Smyth (6) and confirmed in the previous paper (5), the intermediate pituitary of the rat contains a complex mixture of P-endorphin-related molecules. To determine whether cultured rat intermediate pituitary cells synthsized the same forms of p-endorphin observed in extracts of pituitary tissue, cells maintained in culture for 7 days were incubated with [3H]tyrosine for an additional 7 days and radiolabeled P-endorphin-sized material was prepared by immunoprecipitation and gel filtration. The immunoactive p-endorphin-sized material from an extract of rat intermediate pituitary was added to the radiolabeled P-endorphin-sized material and the sample was fractionated by chromatography on a column of SP-Sephadex. As shown in Fig. 1, a peak of radiolabeled @-endorphin co-migrated with each of the major forms of immunoactive p-endorphin present. Thus, even after a total of 14 days in tissue culture, intermediate pituitary cells synthesize molecules similar to the forms of ,B- endorphin found in pituitary tissue extracts.

Kinetics of Biosynthesis In order to determine the time course of labeling of the

various forms of P-endorphin, intermediate pituitary cells were incubated with a labeled amino acid (tyrosine or phenylalanine) for various periods of time, extracted, and immunoprecipitable P-endorphin-sized material was analyzed by SP-Sephadex chromatography. In the experiment shown in Fig. 2, cells were incubated in [‘Hltyrosine for 6 h or 48 h. As shown in the upper panel, the amount of labeled PLPH present at 6 h and at 48 h was the same; the amount of labeled P-endorphin-sized material increased &fold between 6 h and 48 h. The labeled P-endorphin-sized material present after 6 h and 48 h was analyzed by SP-Sephadex column chromatography (Fig. 2, bottom). After 6 h, the major form of labeled P-endorphin eo-migrated with &endorphin( 1-31). The amount of labeled P-endorphin co-migrating with &-endorphin(1-31) did not increase between 6 h and 48 h. The 6-h sample contained a significant peak of labeled P-endorphin at

Portions of this paper (including “Methods” and Figs. 3, 6, and 8 ) 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 Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20014. Request Document No. 80M-2312, cite author(@, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

5689

5690 Further Processing of P-Endorphin in Intermediate Pituitary

observed after SP-Sephadex chromatography were similar to 0 those present at the end of the 3-h pulse labeling period (Fig.

4, top). After the 24-h chase incubation in unlabeled medium, 8o radioactivity co-migrating with &endorphin( 1-31) decreased

to less than 15% of the value observed after 3 h of chase (Fig. 'O Z 4, bottom). The amount of labeled P-endorphin eluting at 0.31 60 f M NaCl doubled between 3 h and 10 h of chase and remained

.r' approximately constant for the duration of the 24-h chase 5 incubation. The amount of labeled P-endorphin eluting at 0.18 4 M NaCl and 0.22 M NaCl increased progressively throughout

300

250

- L 5 200

s \ 4 the entire chase period. The data from the SP-Sephadex 5 I50 30 4- analyses shown in Fig. 4 are summarized in Fig. 3B. The fact

* 100 20 kinetics of labeling shown in Fig. 3B is further evidence that

50 lo are not simply artifacts of the extraction and separation

U

r that analysis of a pulse-chase experiment gives rise to the

the multiple peaks of rat intermediate pituitary /I-endorphin

20 procedures used. 0 20 4 0 60 80 100

FRACTION NUMBER Identification of Forms of P-Endorphin in Intermediate FIG. 1. Analysis of biosynthetically labeled intermediate pi- Pituitary

tuitary 8-endorphin-sized material. Intermediate pituitary cells intact ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ~ ~ d i ~ ~ to Zakarian and Smyth (6) , were prepared from 450-g male rats; 0.3 of a lobe (0.3 nmol of /3- endorphin/well) was plated into each Linbro well in medium contain- the forms Of P-endorphin at 0'18 NaC1 and 0.22 ins 5% adult rat serum. After 7 days in culture, cells in one well were NaC1 be a-N-acety1-P-endorphin(1-27) and P-endor- labeled for an additional 7 days in medium containing ["Hltyrosine (6 Ci/mmol) and then extracted. The radiolabeled material was fractionated by gel fitration, material the size of P-endorphin was pooled, and P-endorphin-related material was prepared by immunoprecipitation; a fraction of the labeled P-endorphin-sized material (0.066 nmol; 70,000 cpm) was mixed with one-half of the 0-endorphin- sized pool from a 570-g male rat intermediate/posterior pituitary extract (0.68 nmol of unlabeled P-endorphin) and the sample was analyzed by SP-Sephadex chromatography. Aliquots (50 pl/fraction) were immunoassayed and liquid scintillation counted. Recovery of radioactivity and immunoactivity was greater than 93%.

8 - 0 BLPH TCA

8-endorphm - 10 8 - 0 BLPH

TCA

8-endorphm - 10

4

fraction 60; the amount of labeled p-endorphin eluting at this position increased 8-fold between 6 h and 48 h. In addition, the 48-h sample contained significant amounts of labeled p- endorphin eluting at lower salt concentrations (fractions 40 and 46). A similar time course of labeling was observed in two other steady labeling experiments.

The precursor-product relationship of these various forms of p-endorphin was further investigated by incubating cells in ["Hlphenylalanine for 3 h (pulse) followed by further incubation in unlabeled medium (chase) for up to 24 h (Figs. 3 and 4). The &endorphin immunoprecipitates for each time point were dissociated and fractionated on a Sephadex G-75 column. The amount of labeled pro-ACTH/endorphin, PLPH, and P- endorphin-sized material present is plotted in Fig. 3A; in order to compare the amount of each form on a molar basis, the amount of radioactivity in each peak was normalized by dividing by the number of phenylalanine residues in that molecule. After the 3 h pulse incubation in [3H]phenylalanine, the extract contained significant amounts of labeled pro- ACTH/endorphin, PLPH, and P-endorphin-sized material. During the subsequent chase incubation in unlabeled medium, the labeled pro-ACTH/endorphin and PLPH were efficiently converted (103%) into labeled P-endorphin-sized material. At the end of the 24 h chase in unlabeled medium, only 7% of the labeled P-endorphin had appeared in the medium.

The SP-Sephadex analyses of each of the pools of labeled ,8-endorphin-sized material are shown in Fig. 4. At the end of the 3-h pulse incubation in ['Hlphenylalanine, the major peak of labeled material (0.37 M NaC1) co-migrated with &endorphin(1-31); a significant peak (27%) of labeled material eluted at 0.31 M NaC1. After the Fist 3 h of chase incubation in unlabeled medium, the amount of labeled P-endorphin-sized material increased (Fig. 3A); the forms of labeled @-endorphin

TIME (hours)

12

FRACTION NUMBER

FIG. 2. Steady labeling of intermediate pituitary cells. Two 400-g male rat intermediate/posterior pituitaries were dissociated equal aliquots of cells were incubated for 6 h or 48 h in 0.4 ml of medium containing ['Hltyrosine (16.7 Ci/mmol). The cell extracts were immunoprecipitated and fractionated by gel filtration. The peaks of radioactivity migrating with mouse tumor cell PLPH and P-endorphin were quantitated by scintillation counting aliquots of each fraction. These data along with the amount of trichloroacetic acid- precipitable radioactivity at each time point are plotted in the upper panel. In the lower panel, the P-endorphin-sized material was pooled and a fraction of it (corresponding to 3.7% of the extract) was analyzed by SP-Sephadex chromatography with unlabeled /3,-endorphin(l-31) as an internal standard. In this analysis, the linear NaCl gradient began at zero NaCl (fraction 6) and ended at 0.6 M NaCl (fraction 105).

Further Processing of @-Endorphin in Intermediate Pituitary 5691

'' OChbse ' ' 0 3 h Chase

FRACTION NUMBER

FIG. 4. Pulse-chase incubation of intermediate pituitary cells: SP-Sephadex analyses. The P-endorphin-sized pools from the experiment described in Fig. 3 were analyzed by SP-Sephadex chromatography with PL-endorphin(l-31) and human P-endorphin( 1- 27) (not indicated) as internal standards (17% of each pool was analyzed). The cell extract after the 3 h of pulse labeling contained 1.14 X IOfi acid-precipitable cpm; after 24 h of chase, the extract contained 0.69 X 10' acid-precipitable cpm.

phin(1-27), respectively. Our preliminary studies were not in agreement with this identification; according to our analyses, the NHz-terminal tyrosine residue in both of these pools of P-endorphin was acetylated. In order to resolve this discrep- ancy, we analyzed each of the major forms of intermediate pituitary ,&endorphin (0.18 M NaC1,0.22 M NaC1,0.31 M NaCl, and 0.37 M NaCl) by rechromatography on SP-Sephadex, gel fdtration on Sephadex G-50, and digestion with chymotrypsin, pronase, and trypsin. Biosynthetically labeled forms of P- endorphin were prepared by preparative SP-Sephadex chromatography. The [3H]tyrosine-labeled material analyzed in Figs. 5 through 8 was derived from the 7-day and 48-h incubations shown in Figs. 1 and 2. Similar results were obtained with several other preparations of labeled ,&endorphin.

Each of the major peaks of labeled P-endorphin was reanalyzed by SP-Sephadex column chromatography (Fig. 5 ) ; each peak eluted at the same salt concentration during reanalysis as during the original analysis. Thus, chromatography on SP- Sephadex in 50% acetic acid does not create the multiple forms of P-endorphin observed. The peak of labeled P-endorphin at 0.18 M NaCl eluted from the column before the a-N- acetyl-Pc-endorphin(1-27) marker (Fig. 5 A ) . This elution position is consistent with the peptide in the 0.18 M NaCl pool having one less positive charge than a-N-acetyl-P,-endorphin(1-27) ( 5 ) . The peak of labeled P-endorphin eluting at 0.22 M NaCl co-migrated with a-N-acetyl-PC-endorphin( 1-27) and not with P,-endorphin(l-27). The peaks of labeled P- endorphin eluting at 0.31 M NaCl and 0.37 M NaCl co-migrated with a-N-acetyl-P,-endorphin(1-31) and P,-endorphin(l-31),

respectively (Fig. 5, B and C ) . For these two peaks, the ["HI- tyrosine-labeled material eluted slightly (0.005 to 0.010 M NaC1) before the synthetic &endorphin markers. This obser- vation could be accounted for by the fact that essentially all of the biosynthetically labeled molecules contained an oxidized methionine residue after the initial SP-Sephadex chromatography and desalting procedures (5). There was no major peak of [3H]tyrosine-labeled P-endorphin that co-migrated with &endorphin(l-27).

The pools of ['Hltyrosine-labeled P-endorphin were analyzed by gel filtration in 6 M guanidine HCl in the presence of synthetic marker peptides. This column is capable of resolving P-endorphin(1-31) from &endorphin( 1-27) (5). As expected, human and camel P-endorphin elute at the same position on this column. Each peak of labeled P-endorphin was analyzed in the presence of P,-endorphin(1-31) and glucagon (Fig. 6 ) ; in addition, each peak of labeled P-endorphin was analyzed in the presence of other appropriate P-endorphin standards (data not shown). The peak of labeled P-endorphin eluting at 0.18 M NaCl contained molecules clearly smaller than P-endorphin(1-31) and eluted at about the position of P-endorphin(1- 27) (Fig. 6 A ) . The peak of labeled P-endorphin eluting at 0.22 M NaCl eluted with or slightly before a-N-acetyl-&endorphin(1-27) (Fig. 6B). The peak of labeled P-endorphin eluting at 0.31 M NaCl eluted slightly earlier than P,-endorphin(l-31) (Fig. 6C); when analyzed with a-N-acetyl-P,-endorphin(l-31) as the internal marker, the labeled ,&endorphin and immunoactive P-endorphin eluted together. The peak of labeled P- endorphin eluting at 0.37 M NaCl eluted with &endorphin( 1- 31) (Fig. 6D). Thus, the 0.18 M and 0.22 M NaCl peaks were in the size range of P-endorphin(1-27), while the 0.31 M and 0.37 M NaCl peaks were in the size range of P-endorphin( 1-31).

Enzymatic Digests-Smyth et al. (7) identified a-N-acetyltyrosine as the NH2 terminus of some forms of P-endorphin in pig pituitary. Therefore, the [3H]tyrosine-labeled forms of P- endorphin from rat intermediate pituitary cultures were digested with pronase and/or chymotrypsin to liberate labeled tyrosine or a modified form of labeled tyrosine. Digestion of the peak of material eluting at 0.37 M NaCl with pronase yielded [3H]tyrosine based on paper chromatography (Fig.

A.O.18MNaCI

I50

I00

5 0 - - L

9 0 .- ,= Q c - 0 0

4 300 a 0 0

E

and0.31MNoCl N-acrtyl-B~-rndorphin(l-27) U

300 f Q

200 200 - Q

.-

0 100 100 0 - r L O

300

! E .-

150

,OOt I00

0 20 40 60 80 100

FRACTION NUMBER

FIG. 5. Reanalysis on SP-Sephadex. The pools of ['Hltyrosine- labeled rat intermediate pituitary P-endorphin indicated were mixed with synthetic /?-endorphin markers and reanalyzed by SP-Sephadex chromatography. Aliquots (100pl/fraction) were immunoassayed and the remainder of each fraction was liquid scintillation counted.

5692 Further Processing of ,&Endorphin in Intermediate Pituitary

7A) and paper electrophoresis (not shown); in both separation systems, over 92% of the label co-migrated with tyrosine. Digestion of the peaks of material eluting at 0.18 M, 0.22 M, and 0.31 M NaCI with pronase yielded a mixture of products, none of which co-migrated with tyrosine; subsequent digestion with carboxypeptidase Y did not simplify the pattern observed. Digestion of each of these peaks with chymotrypsin yielded a single peak of 'H-labeled material that co-migrated with a-N-acetyltyrosine during paper chromatography (Fig. 7A), paper electrophoresis (Fig. 7B), and high performance liquid chromatography (not shown). The high performance liquid chromatography system used was capable of resolving a-N-acetyl-tyrosine from a-N-acetyl-0-methyltyrosine and a-N-,0-diacetyltyrosine; for each of the peaks (0.18 M, 0.22 M, and 0.31 M NaCl), greater than 90% of the label eluted at the position of a-N-acetyltyrosine and no labeled a-N-,0- diacetyltyrosine was detected (<0.5% of the total counts).

The various forms of [3H]tyrosine-labeled /I-endorphin obtained by SP-Sephadex chromatography were treated with dimethyl sulfoxide to ensure that all of the methionine residues were oxidized. Tryptic digests were analyzed by paper chromatography (Fig. 8A ) and paper electrophoresis (Fig. 8, B and C ) . The material in each pool of labeled P-endorphin generated a single major labeled tryptic peptide. The same ["Hltyrosine-containing tryptic peptide was observed in digests of the material eluting at 0.18 M, 0.22 M, and 0.31 M NaCI; the labeled tryptic peptide co-migrated with oxidized a-N-acetyl-P-endorphin(1-9) during paper chromatography and had the charge expected for a-N-acetyl-/3-endorphin(1-9) at pH 3.5 and at pH 6.35. The ["Hltyrosine-containing tryptic peptide generated from the material eluting at 0.37 M NaCl co-migrated with oxidized P,-endorphin(l-g) in each of the separation systems. When reduced with ethyl methyl sulfide and then digested with trypsin, the material eluting at 0.22 M NaCl and 0.31 M NaCl produced primarily (265%) a single labeled peptide that co-migrated with a-N-acetyl-P,-endorphin( 1-9) during paper chromatography. Thus, based on analysis of chymotrypsin/pronase and trypsin digestion products,

400 I I 1 I

A. PC 0.31MNoCI

3 0 0 - oO.37MNaCI

4 0.22 MNaCl 0.31 MNaCl

10 20 30 40

SLICE NUMBER

FIG. 7. Characterization of NHz-terminal tyrosyl residues. The pools of [3H]tyrosine-labeled intermediate pituitary P-endorphin indicated were digested with chymotrypsin alone (0.22 M NaCl and 0.31 M NaCl) or chymotrypsin followed by pronase (0.37 M NaC1) and analyzed by descending paper chromatography ( A ) or pH 6.35 paper electrophoresis ( B ) . Tyrosine and a-N-acetyltyrosine were analyzed at the same time.

c f-N-Ac-Bc-ondo(l-27)

FRACTION NUMBER

FIG. 9. [35S]methionine-, [3H]histidine-labeled intermediate pituitary/3-endorphin-sized material. Intermediate pituitary cells were prepared from two 250-g male rats. The entire cell suspension was incubated in 0.47 ml of medium containing [35S]methionine and ['Hlhistidine for 70 h, followed by a 2-h chase in 2 ml of unlabeled complete medium. Cells were extracted and immunoprecipitable /3- endorphin-sized material was fractionated by SP-Sephadex chromatography; a-N-acetyl-fl,-endorphin(l-27), a-N-acetyl-&endorphin- (1-31), and /3c-endorphin(l-31) were included. Aliquots of fractions (100 pl) were liquid scintillation counted and spillover of 35S into the 3H channel (0.38 of the higher energy channel number) was corrected; background radioactivity (20 cpm in each channel) has been sub- tracted.

the NH2 terminus of the material eluting at 0.37 M NaCl was similar or identical with that of unmodified /3-endorphin, while the NH2-terminal region of each of the peaks eluting at a lower NaCl concentration was simiiar or identical with that of a-N-acetyl-P-endorphin.

Taking into account the ion exchange and gel filtration analyses of the intact cell products and the analyses of proteolytic digests, one can identify three of the forms of p- endorphin found in rat intermediate pituitary. The peak of material eluting from the SP-Sephadex column at 0.37 M NaCl is P-endorphin(1-31); the peak at 0.31 M NaCl is a-N-acetyl- P-endorphin(1-31); and the peak at 0.22 M NaCl is rY-N-acetyl- P-endorphin(1-27).

Identification of Material Eluting from the SP-Sephadex Column a t 0.18 M NaCl

The NH2-terminal region of the material eluting from the SP-Sephadex colum at 0.18 M NaCl was indistinguishable from the NHn-terminal region of a-N-acetyl-pc-endorphin(l- 27). However, the peak of material eluting at 0.18 M NaCl differed from a-N-acetyl-P,-endorphin( 1-27) in several re- spects. It eluted as though it had one less positive charge during chromatography on SP-Sephadex (Fig. 5); it appeared to be slightly smaller during gel fitration (Fig. 6); and it became labeled later than a-N-acetyl-P,-endorphin( 1-27) during biosynthesis (Figs. 2-4). All of these properties suggested that the material eluting at 0.18 M NaCl could be a-N-acetyl- /3-endorphin(1-26). Rat P-endorphin contains Met' and His" (3, 16); in order to detect /3-endorphin-related molecules shorter than P-endorphin(l-27), intermediate pituitary cells were incubated in medium containing [3H]histidine and ["'S]methionine and the immunoprecipitable P-endorphin was fractionated by SP-Sephadex chromatography (Fig. 9). As expected, peaks of [35S]methionine-labeled P-endorphin appeared at 0.18, 0.22, 0.31, and 0.37 M NaC1. The material eluting at 0.22, 0.31, and 0.37 M NaCl was also labeled with ["]histidine; the ["Hlhistidine-labeled material co-migrated with a-N-acetyl-P,-endorphin(l-27), a-N-acetyl-&endor-

Further Processing of j3-Endorphin in Intermediate Pituitary

ACETYLATION

5693

Acetyl

Acetyl

Acetyl

.yS-Gly - G ~ n 3 1

FIG. 10. Post-translational processing of j?-endorphin in rat intermediate pituitary.

phin(l-31), and &endorphin( 1-31), respectively. For each of these three pools of material, the ratio of [3H]histidine to [35S]methionine radioactivity (counts per minute/cpm) was 12.6 -+ 0.7. The material eluting at 0.18 M NaCl was not significantly labeled with [3H]histidine; the ratio of [3H]histidine to [35S]methionine radioactivity in the pool of material eluting at 0.18 M NaCl was 60-fold lower than the same ratio for the pools of material eluting at 0.22,0.31, and 0.37 M NaC1.

DISCUSSION

The multiple forms of P-endorphin found in rat intermediate pituitary extracts are synthesized by intermediate pituitary cell suspensions and by longer term intermediate pituitary cultures. Thus, primary cultures of rat anterior and intermediate pituitary display at least some of the differences in P-endorphin metabolism seen in vivo (5). Intermediate pituitary cells in culture have been removed from the inner- vation they normally receive in uiuo (17,18); in many tissues, for example, in skeletal muscle (19) and in the pineal (20), denervation leads to rapid alterations in tissue characteristics. Further experiments will be required to determine whether denervation brings about more subtle changes in intermediate pituitary metab~lism.~

The P-endorphin-sized material in the rat intermediate pituitary was resolved into four major components: P-endorphin(1-31) (0.37 M NaCI), a-N-acetyl-P-endorphin(1-31) (0.31 M NaCl), a-N-acetyl-P-endorphin(1-27) (0.22 M NaCl), and

The data presented in Fig. 1 suggest that there may be important changes in the rate or extent of the post-translational modifications performed by denervated intermediate pituitary c e k , the pattern of labeled 8-endorphin-sized material present after 7 days of incubation with [3H]tyrosine did not quantitatively match the pattern of immunoactive P-endorphin found in extracts of intermediate pituitary tissue. On the other hand, the lack of quantitative agreement between the labeled and immunoactive molecules in Fig. 1 could be a result of the low turnover rate of these peptides in culture. Intermediate pituitary cells secrete only 7% of their cellular hormone content per day (Fig. 3A); at this rate, only one-half of the tissue 8-endorphin would be secreted in a week in culture (as in Fig. 1). The turnover rates for insulin in cultured pancreatic islets and cathepsin D in cultured kidney cells are also on the order of days (21,22). Certainly, the absolute rate of production of 8-endorphin by the intermediate pituitary cultures is similar to the production rates for other proteins in culture (21, 23). Assuming equilibration of the [3H]tyrosine in the medium with the pool of tyrosine used for protein synthesis (if incorrect, the calculated rate will be an underestimate; discussed in detail in Refs. 23 and 24), and using 50,OOO cells/intermediate pituitary, one can calculate from the 6-h time point in Fig. 2 that the intermediate pituitary cells were producing about 4 X 10' molecules of pro-ACTH/endorphin/cell/h. A comparable calculation by Breul et al. (23) indicated that human diploid fibroblasts produced 0.07 X 10' molecules of procollagen/cell/h (procollagen is about 10 times larger than pro-ACTH/endorphin). Rat pancreatic islets (21) produced 16 X 10' molecules of proinsulin/cell/h (proinsulin is one-thud as large as pro-ACTH/endorphin).

a-N-acetyl-P-endorphin(1-26) (0.18 M NaC1). The forms of /?-endorphin were identified by comparing biosynthetically labeled molecules to synthetic P-endorphin standards and by analyzing proteolytic digests of the labeled forms of P-endorphin (Figs. 5-8). The peak of material at 0.18 M NaCl could contain more than one component since chromatography on SP-Sephadex in 50% acetic acid would not be expected to resolve molecules that differed only by the presence or absence of Alaz6. A carboxypeptidase B-like activity is thought to be important in the proteolytic processing of many prohor- mones including pro-ACTH/endorphin (25-28); carboxypeptidase B is known to remove both Lys and His from a hemo- globin peptide ending with the same sequence as P-endorphin(26-28) (Alaz6-His27-Lys28) (29). Thus, all of these forms of P-endorphin could be generated by the action of the trypsin- like and carboxypeptidase B-like enzymes already believed to be involved in the post-translational processing of prohor- mones.

Rudman et al. (30) detected two forms of a-melanocyte- stimulating hormone in pituitaries from several species including rat. In one form, the NH2-terminal serine residue was a-N-acetylated and in the other form it was N,O-diacetylated. No N,O-diacetyltyrosine was detected in any of the forms of rat /%endorphin. Tyrosine sulfate occurs in cholecystokinin in a sequence similar to that of P-endorphin(1-5) (31). Based on incubation of intermediate pituitary cells with Hi3'S04 and on pH 10 paper electrophoresis of a pronase digest of [3H]tyrosine-labeled P-endorphin, no evidence for the presence of tyrosine sulfate in rat intermediate pituitary &endorphin was found.

In this study and in that of Zakarian and Smyth (6), the peaks of rat intermediate pituitary P-endorphin eluting at 0.31 M and 0.37 M NaCl were identified as a-N-acetyl-B-endorphin(1-31) and P-endorphin(1-31), respectively. Zakarian and Smyth (6) identified the peak of material eluting at 0.22 M NaCl as P-endorphin(1-27) based on comparison to the elution position of pig pituitary P-endorphin-sized material. In our hands, synthetic /3,-endorphin(l-27) eluted from the SP-Seph- adex column at 0.28 M NaC1, while synthetic a-N-acetyl+,- endorphin(1-27) eluted at 0.22 M NaCl. Furthermore, in our studies, the p-endorphin eluting from the SP-Sephadex column at 0.22 M NaCl had an acetylated NH:! terminus. Zakarian and Smyth (6) identified the peak of material eluting at 0.18 M NaCl as a-N-acetyl-P-endorphin(1-27); in our hands, the material eluting from the SP-Sephadex column at 0.18 M NaCl had one less positive charge than a-N-acetyl-P,-endorphin(l- 27) and lacked Hisz7.

The biosynthetic pathway leading to the production of the various forms of intermediate pituitary /?-endorphin is summarized in Fig. 10. As shown before, PLPH is a short lived biosynthetic intermediate in the conversion of pro-ACTH/

5694 Further Processing of /3-Endorphin in Intermediate Pituitary

endorphin into P-endorphin-sized material (9, 14). Cells were incubated with labeled amino acid for 3 h (Figs. 3 and 4); at this time, 70% of the labeled P-endorphin-sized material was P-endorphin( 1-31) and 30% was a-N-acetyl-P-endorphin(1- 31). Two separate lines of evidence suggest that P-endorphin/ 1-31) is a biosynthetic intermediate in the production of the other forms of P-endorphin. First, in the steady labeling experiment shown in Fig. 2, the amount of labeled P-endorphin( 1-31) did not increase between 6 and 48 h of incubation, while label continued to accumulate in each of the other forms of /?-endorphin. Second, the time course shown in Fig. 3B indicates that the P-endorphin(1-31) pool became labeled more rapidly than the other forms of P-endorphin and lost radioactivity more quickly than any of the other forms of P- endorphin. The predominant biosynthetic processing pathway for P-endorphin( 1-31) begins with acetylation of the tyrosine residue at the NH2 terminus to produce a-N-acetyl-P-endorphin(1-31). On a much slower time scale, the COOH-terminal region is shortened to produce a-N-acetyl-P-endorphin(1-27) and eventually a-N-acetyl-P-endorphin( 1-26).

These studies pose several interesting questions for future experiments. Which of the various forms of P-endorphin are secreted in vivo and under what circumstances? Are there physiologically important controls on the post-translational processing of P-endorphin? What biological actions might the various forms of P-endorphin possess? How do anterior pituitary and intermediate pituitary cells produce different product peptides?

Acknowledgments-We thank Diane Honnecke for her expert technical assistance and George Tarver for preparing the drawings. Chris Glembotski and Bob Dores helped to make the manuscript more readable.

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Further Processing of /I-Endorphin in Intermediate Pituitary 5695

30. A . Call extracts on 6-75

A 8- l ipotropin Pro-ACTHIendorphin

8-endorphin-sized 20 ” k 10

8-endo-sired in mediu

-.. 3, + 1 B.Cell/3-endo-sized on SP-Sephadex 1

0 3 10 24

CHASE TIME (hours)

A.O.ISM NaCl

8-endo(l-31)

100

IOOL 8 I t

m= 2001 fl

30 40 50 60 70 0 FRACTION NUMBER

o-N-ocetyl-Bc-endo

Be-endo( l -9 )

10 20 30 40

further analysis of post-translational processing of p-endorphin in

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