THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 257, No. 2. Issue of January 25, pp. 832-838. 1982

Incorporation of Canavanine into Rat Pars Intermedia Proteins Inhibits the Maturation of Pro-opiomelanocortin, the Common Precursor to Adrenocorticotropin and P-Lipotropin*

(Received for publication, August 10, 1981)

Philippe Crinet and Edith Lemieux From the Departement de Biochimie, Uniuersite de Montreal, Montreal, H3C 357 Canada

Pro-opiomelanocortin, the common glycoprotein pre- cursor to adrenocorticotropin and p-lipotropin, is the most abundant protein synthesized in rat nearointer- mediate lobes. Dissected rat neurointermediate lobes were incubated in the presence of canavanine, an ana- log of arginine, to determine (a) whether canavanine could be incorporated into pro-opiomelanocortin mol- ecules and (b) if incorporation occurs, whether there is any effect on the processing mechanism of the prohor- mone. Preincubation of rat neurointermediate lobes for 16 h in the presence of 10 m~ canavanine results in the production of pro-opiomelanocortin molecules in which most, if not all, the arginine residues have been re- placed by canavanine. Identification of canavanine- containing pro-opiomelanocortin forms was done by two-dimensional electrophoresis, tryptic and chymo- tryptic peptide mapping, as well as by analysis, on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the fragments resulting from a partial diges- tion with chymotrypsin. During pulse-chase experi- ments, canavanine-containing pro-opiomelanocortin molecules were found to be processed at a much slower rate than the normal precursor forms: after a 2-h chase, conversion of approximately 25% of the analog-con- taining prohormone was observed compared to 83% of the nonanalog-containing precursors. Moreover, the small proportion of canavanine-containing precursor molecules which had undergone cleavage during the chase yielded atypical large molecular weight peptides. These results indicate that canavanine incorporation into neurointermediate lobe proteins considerably slows down the conversion of pro-opiomelanocortin into its different end products.

It is now well established that many polypeptide hormones (1-7) as well as other proteins or enzymes (8-10) are initially synthesized as larger precursors which are subsequently proc- essed to produce the biologically active products. The initial cleavage event occurs at the site of synthesis of the poly- peptide in the rough endoplasmic reticulum (11) and consists of the co-translational removal of the “signal peptide” located at the NH2 terminus of the protein. In many cases, this initial maturation event is followed by other cleavage processes

* This work was supported by Grant MA6612 from the Medical Research Council of Canada and by grants from the Banting Research Foundation and the Montreal Cancer Research Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a Medical Research Council scholarship.

occurring during sequential segregation and translocation of the hormone within subcellular organelles (12, 13). Evidence derived from the known primary structures of many prohor- mones and precursor proteins indicates that precursor matu- ration could be mediated by a dual enzymic activity combining a trypsin-like endopeptidase and a carboxypeptidase B-like enzyme acting sequentially at the site of pairs of basic amino acid residues (14).

Pro-opiomelanocortin, the common precursor to P-endotro- phin and adrenocorticotropin in the pituitary (15-18), pro- vides a unique model for studying prohormone maturation mechanisms. From the known primary structure of the pre- cursor molecule (18-20), it appears that the polypeptide ini- tially synthesized consists of three units corresponding to each of the two main hormones and the putative hormone destined to be released from the initial polypeptide: ,&lipotropin, ACTH’, and the NH’-terminal peptide (Fig. 1). All three of these units are separated from each other by pairs of basic amino acids. Within each of the three main units, additional pairs of basic amino acid residues can be found flanking conserved melanotropic-like peptides.’ This structure indi- cates that all the main units should be separated from each other during the processing of the precursor. Further cleavage within each unit could also occur to yield smaller melanotro- pic-like peptides and ,&endorphin.

In an effort to understand the role of pairs of basic amino acid residues in the maturation process of POMC, we have tried to incorporate canavanine, an arginine analog, into the precursor molecule. We report here that incorporation of canavanine into rat neurointermediate lobe proteins inhibits conversion of POMC into its different end products. While this study was being completed, similar results were presented by Noe on the inhibition of islet prohormone maturation (21).

EXPERIMENTAL PROCEDURES

Preparation and Intubation of Neurointermediate Lobes-Iso- lated rat neurointermediate lobes were preincubated under an atmos- phere of 95% air-5% COS at 37 “C for 16 h in RPMI 1640 buffer containing 10 mM canavanine instead of arginine. The volume of buffer used for each piece of tissue was 50 p1. Control experiments were carried out using the same preincubation protocol and the same incubation medium but without canavanine. Preliminary experiments had shown that removal of any of the essential amino acids present in RPMI medium had no effect on the rate of protein synthesis, which

’ The abbreviations used are: ACTH, adrenocorticotlopic hormone, adrenocorticotropin; POMC, pro-opiomelanocortin; P-LPH, P-lipo- tropic hormone, P-lipotropin; NaDodSOI, sodium dodecyl sulfate; BSA, bovine serum albumin; MSH, melanocyte-stimulating hormone. ’ In the rat, however, P-melanotropin (P-MSH, residues 41 to 58 of

the bovine P-LPH sequence) is not preceded by a pair of basic amino acids and this hormone should not he produced through the normal processing pathway.

832

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Inhibition of Pro-opiomelanocortin Maturation by Canavanine 833

I . I N - tsrdn'ol $+de ,' ' . p C t H

~~ , . I .

B .'k'P n

, I , , . , . , I ,

I. I

lyrval+glyhtspheang ;ertyr,armergluhirphs.;Q t y r g l y g l y p h e ~ t I h r ~ = r g l u l y ~

FIG. 1. Partial primary strucutre of rat pro-opiomelanocor- tin. The complete amino acid sequence of the molecule was deduced by Drouin and Goodman (19) from the DNA nucleotide sequence. Numbering is from the NH2 terminus of the prohormone lacking its signal sequence as determined by Gossard et al. (38). Only the main features of the molecule are presented the relative positions and sequences of the active "core peptides" and the positions of the pairs of basic amino acid residues.

under these conditions remained linear for more than 24 h. After the preincubation, the pituitary explants were incubated in RPMI me- dium with or without canavanine instead of arginine and containing one of the following radioactive amino acids: ["Hlphenylalanine (104 Ci/mmol), ["]arginine (21 Ci/mmol), or [%]methionine (1100 Ci/ mmol) at the concentration given in the text. In pulse-chase incuba- tions, tissue explants which had previously been preincubated in the presence or in the absence of canavanine were fiist pulse-incubated for 30 min in RPMI containing [3H]phenylalanine (1 mCi/ml), then washed in complete RPMI medium in which the phenylalanine con- centration had been increased to 2 mM, and finally incubated in the same medium for an additional 2-h period (chase incubation). Under those conditions, less than 5% of the labeled synthesis products were recovered in the incubation medium.

Extraction of Pituitary Peptides and Electrophoresis-Radioac- tive neurointerediate lobe peptides were extracted by three cycles of freezing and thawing of the tissue explants in 50 pl of O'Farrell's lysis buffer (22), as explained previously (23). The samples were then centrifuged for 5 min at 12,800 X g in an Eppendorf table-top centrifuge and aliquots of the supernatants were directly layered on top of a cylindrical isoelectric focusing gel containing pH 3.5-10 ampholytes (23). After isoelectric focusing, the gel was layered on top of a 10-1576 acrylamide gradient slab gel containing NaDodS04 and proteins were then separated by electrophoresis in the second dimen- sion as described earlier (23). For one-dimensional polyacrylamide gel electrophoresis, tissues explants were simply boiled for 2 min in Laemmli's sample buffer (24), centrifuged as above, and directly analyzed on a discontinuous 8.4 M urea-polyacrylamide gel as de- scribed by Goldsmith et al. (25).

Peptide Mapping of Neurointermediate Lobe Peptides-Regions of two-dimensional polyacrylamide gels containing radioactive pep- tides were cut out and digested by trypsin as explained earlier (26). Radioactive fragments were analyzed by high voltage paper electro- phoresis a t pH 6.5 according to Roberts and Herbert (16). Partial chymotrypsin digestion of radioactive proteins was performed on pieces cut from a two-dimensional gel as described by Cleveland et al. (27) and the fragments were resolved by electrophoresis on a gradient polyacrylamide slab gel as explained previously (23).

In Vitro Tqpsin Digestion of POMC-Neurointermediate lobes which had been preincubated in the presence or in the absence of 10 mM canavanine for 16 h were then incubated for 1 h with methionine (Amersham, 1450 Ci/mmol; 5 Ci/ml) and the radioactive proteins were analyzed by two-dimensional gel electrophoresis. Ra- dioactive protein spots corresponding to precursor forms were cut from the gels, and the proteins were electroeluted according to the procedure of Allington et at. (28). The samples were then lyophilized, redissolved in 50 pl of 0.1 M NH2HCOs, pH 8.0, together with 60 pg of carrier BSA. To half of each sample, 0.03 pg of trypsin was added and incubation was carried out for 0, 30, and 60 min at 37 "C. The other half of the sample was incubated without trypsin to verify the absence of endogenous or contaminating protease activity in the samples. Trypsin digestion was stopped by lyophilization and the samples were prepared for electrophoresis in 10% acrylamide/NaDodS04 rod gels according to Weber and Osborn (29). After electrophoresis, the gels were cut into 2-mm slices and the proteins were eluted by incubation at 37 "C for 16 h in 0.1% NaDodSO4, 0.5 M urea, 5 mM NaHC03. Radioactivity was counted after the addition of Triton-toluene scin- tillation mixture to each fraction.

Miscellaneous Procedures-Radioactive amino acid incorporation

into trichloroacetic acid-precipitable proteins was determined accord- ing to Ivarie and OFarrell (30). Protein concentration was measured by the method of Bradford (37). Autoradiography and fluorography of dried slab gels were performed as described earlier (23). When different amounts of radioactive proteins were analyzed, the time of exposure was adjusted so as to keep constant the product of the number of counts/min by the time of exposure.

Materials-Canavanine was purchased from Sigma. RPMI buffer as a Selectamine kit was from Gibco. All radioactive amino acids were bought from New England Nuclear except one batch of ["'S]methio- nine which came from Amersham. All the reagents for isoelectrofo- cusing were from Bio-Rad. Acrylamide, urea, and NaDodSOl for electrophoresis were bought from British Drug House. Trypsin (L-1- tosylamide-2-phenylethyl chloromethyl ketone-treated) and chymo- trypsin (both from Worthington) were dissolved in 0.1 M NH,HCO,I, pH 8.0, buffer immediately before use at a concentration of 1 mg/ml and diluted at the appropriate concentration. All other chemicals were of the purest grade available.

RESULTS

Incubation of Neurointermediate Lobes in the Presence of Canavanine-In order to ensure that canavanine was able to compete with arginine for protein synthesis during incubation of rat neurointermediate lobes with radioactive amino acids, we measured the rate of ["Hlarginine incorporation into tri- chloroacetic acid-precipitable material in the presence or in the absence of canavanine. As shown in Fig. 2B, preincubation of rat neurointermediate lobes for 16 h in the presence of 10 mM canavanine decreased the rate of ["]arginine incorpora- tion into proteins by 75%. However, as shown in Fig. 2A, the overall rate of protein synthesis was itself reduced by 45% as judged from the rate of ["'S]methionine incorporation into trichloroacetic acid-precipitable material. Attempts to dimin- ish this nonspecific effect of canavanine on protein synthesis by reducing the preincubation time or by lowering the analog concentration also resulted in a decrease of the ability of the analog to inhibit ['3H]arginine incorporation into proteins. Therefore, a protocol consisting of a 16 h preincubation in the presence of 10 mM canavanine was adopted for all the subse- quent experiments.

Characterization of POMC Forms Synthesized in the Pres- ence of Canavanine-In order to verify that arginine residues had been replaced by canavanine in protein synthesized dur- ing the incubation of the pituitary explants with radioactive amino acids, we analyzed the radioactive proteins by two- dimensional gel electrophoresis, a method which has already proven very efficient for resolving intermediate lobe proteins (23). Since the guanido group in canavanine is much less basic than in arginine (its pK, is 9.25 in canavanine as compared to 12.48 in arginine (31)), replacement of arginine residues by canavanine should lower the isoelectric point of a protein and therefore shift its position toward the acidic region of isoelec- tric focusing gels without affecting its molecular weight.

When analyzed by two-dimensional gel electrophoresis, [:'HI phenylalanine-labeled POMC molecules can be resolved into a series of spots (arrowheads on Fig. 3A) with PI values ranging from 6.0 to 8.5. All these proteins had previously been identified as various forms of POMC by peptide mapping (23). In the presence of canavanine, numerous alterations in the protein pattern were observed: in the acidic region of the gel, several proteins migrating with M , 50,000 had disappeared and the synthesis of others dramatically increased. On the other hand, all the spots corresponding to various forms of POMC had disappeared while new proteins migrating with approximately the same molecular weight (arrowheads in Fig. 3B) were observed. This latter class of proteins could either be new protein induced in intermediate lobe cells in the presence of the analog, as already observed in primary cultures of chicken fibroblasts (321, or they could correspond to POMC

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834 Inhibition of Pro-opiomelanocortin Maturation by Canavanine

A (%) METHIONINE

I 1

10 -

5 -

- 0 0 0 1 2 3 4 5 6 7 8 9

0 1 2 3 4 5 6 7 8

1IMf (h)

FIG. 2. Rate of [35S]methionine and [3H]arginine incorpora- tion into rat intermediate lobe proteins in the presence or in the absence of canavanine. Hat neurointermediate lobes were preincubated individually for 16 h in 50 pl of HPMI incubation medium lacking arginine and methionine and containing or not con- taining 10 mM canavanine. The incubation medium was then supple- mented with [”S]methionine (1100 Ci/mmol, 200 pCi/ml) and [“HI arginine (21 Ci/mmol, 714 pCi/ml), and the incubation proceeded for up to 9 more hours. Incorporation of radioactive amino acids into trichloroacetic acid-precipitable material and protein content of tissue extracts were then measured as described under “Experimental Pro- cedures.” The number of disintegrations/min in each sample was computed taking into account the spilling over of one channel into the other. Each point is the mean obtained from duplicate determi- nations performed on two lobes incubated separately. A, [’%]methi- onine incorporation; B, [:‘H]arginine incorporation.

forms where part or all of the arginine residues have been substituted for canavanine. In order to distinguish between these two possibilities, peptide maps of these two different sets of proteins were developed. [:”SS]Methionine-labeled pre- cursor forms (marked I , 11, and 111 on Fig 3A) were excised from the gel and digested with trypsin and the fragments were analyzed by high voltage paper electrophoresis. All three of these POMC forms exhibited the pattern shown in Fig. 4A for form I. The three peaks of this pattern were shown to co- migrate with the three methionine-containing tryptic frag- ments of POMC used as standard: a neutral peptide co-mi- grating with synthetic /3-LPH (61-69), a positively charged peptide corresponding to ACTH (1-8), and the region corre- sponding to yMSH (1-7) which in this system migrates as a double peak, as already observed by others (33). When pep-

* 94 67

c 4 3

30

e 2c

c 94 C 6 7

e 4 3

e 30

c 20

FIG. 3. Two-dimensional gel electrophoresis of [3H]phenyl- alanine-labeled POMC molecules containing or not containing canavanine residues. After a preincubation of 16 h in the presence ( B ) or in the absence ( A ) of canavanine (final concentration, 10 mM), each rat neurointermediate lobe was pulse-incubated in a medium containing or lacking canavanine supplemented with [’Hlphenglala- nine (104 Ci/mmol, 1 mCi/ml). Aliquots of tissue extracts in O’Farrell’s lysis buffer (see “Experimental Procedures”) containing 72,000 cpm ( A ) or 41,000 cpm ( B ) were analyzed by two-dimensional gel electrophoresis. Exposure time was 3 days in A and 5 days in B. ZEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis.

tides I’ and 11’ (Fig. 3B) synthesized in the presence of cana- vanine were digested with trypsin and the fragments analyzed by high voltage paper electrophoresis, two identical patterns were obtained, both completely different from the map of the [:‘“S]methionine-labeled precursor obtained previously (only the pattern obtained with peptide I’ is presented in Fig. 4A). 1) peaks N and N’ characteristic of the NHZ-terminal [“‘S] methionine-labeled tryptic fragments had disappeared; 2) a new peak migrating with RF = 0.18 was observed while the peak co-migrating with ACTH (1-8) greatly diminished; 3) a larger proportion of peptides remained at the origin (probably as large fragments).

All these observations, however, could be predicted assum- ing that failure to cleave the protein with trypsin a t positions where arginine has been replaced by canavanine will result in the following modifications of the tryptic fragments: 1) the y- MSH (1-7) peptide will be included in a larger sequence from positions 50 to 96 in the rat POMC sequence (19) and will contain several canavanine residues; 2) ACTH (1-8) will be preceded by a canavanine residue (from the Lys-Arg doublet separating the NHZ-terminal sequence of the ACTH moiety) and will be extended to the lysine residue in position 11 of the ACTH sequence, including a canavanine residue in position 8; 3) the /3-LPH (61-69) fragment will contain an additional canavanine residue (replacing the Arg residue in position 60

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Inhibition of Pro-opiomelanocortin Maturation by Canavanine 835

A

I I , I

8MStI (1-7) : : 1 :

I a0.75 a0.50 *0.25 0 -025 -0.50 -0.75 -1.00

8

, -0.75 -0.50 -0.25 0 -0.25 - C 50 -075 IO0 MOBILITY

FIG. 4. High voltage paper electrophoresis of the [3sS]methi- onine-labeled tryptic ( A ) and chymotryptic (B) fragments of POMC molecules containing or not containing canavanine residues. A. [""S]methionine-labeled precursor forms synthesized in the presence or in the absence of 10 mM canavanine were resolved by two-dimensional gel electrophoresis as shown in Fig. 3. [""SIMethio- nine-labeled proteins spots were revealed by autoradiography and those corresponding to forms I, 11, and 111 (as in Fig. 3A) or to forms I' and 11' (as in Fig. 3B) were digested with trypsin (in the presence of 50 pg of carrier BSA) for 16 h at 37 "C (final trypsin to BSA ratio, 1/10). The fragments eluting out of the gel pieces were lyophilized, oxidized with performic acid, and analyzed by high voltage paper electrophoresis a t pH 6.35 on Whatman 3 MM paper. After 4 h of electrophoresis at 1.ooO V, the paper strips were cut into 0.5-cm bands and soaked in 0.12 N HCI, and the radioactivity was counted. Syn- thetic peptides (ACTH 1-8, a-MSH 1-7. and P-LPH 61-69, generous gifts from Dr. Michel Chretien. Clinical Research Institute of Mon- treal) were run in parallel as standards. H p values were computed relative to lysine ( H F I . ~ . = 1) and the R p of glycine was taken as 0. Since the patterns obtained with forms I, 11, and 111 in one case and I' and 11' in the other were all identical, only the results of form I and I' are presented here. B, oxidized tryptic peptides from A were further digested for 6 h with chymotrypsin (final enzyme-to-substrate ratio, 1/10) and the resulting tryptic/chymotryptic-labeled fragments were

of P-LPH) at its NH2 terminus. By further digestion of the tryptic peptides from the [:"S]methionine-labeled POMC mol- ecules with chymotrypsin, it is, however, possible to generate labeled fragments not containing arginine residues. Therefore, if proteins I' and 11' correspond to forms of POMC containing canavanine instead of arginine, digestion of the ["'S]methio- nine-labeled tryptic fragments with chymotrypsin should pro- duce tryptic/chymotryptic peptides identical with those of the normal precursor forms. Fig. 4B shows that when tryptic peptides obtained from both sets of molecules were further digested with chymotrypsin, very similar high voltage paper electrophoresis maps were obtained. Although no attempts were made to identify the resulting tryptic/chymotryptic frag- ments, these results provide the first evidence that the new acidic proteins synthesized in the presence of canavanine represent altered forms of POMC.

To confirm this conclusion, additional proof of the identity of these proteins was obtained through partial digestion of the sets of proteins with chymotrypsin and analysis of the frag- ments by polyacrylamide gel electrophoresis in the presence of NaDodSO,. Since chymotrypsin should not be affected by the presence of canavanine in the molecule and since the digestion products are resolved according to their size and not their charge, there should be complete identity between the two patterns, as was indeed observed in Fig. 5. The combined results obtained from the two methods of peptide mapping demonstrate that the acidic proteins synthesized in the pres- ence of canavanine represent forms of POMC where most, if not all, of the arginine residues have been replaced by cana- vanine.

Maturation of POMC Forms Containing Canauanine- Previous studies have shown that when a pulse incubation performed on neurointermediate lobes was limited to 30 min the most predominant radioactive proteins had apparent M,

CONT

-.

a

lir

P d

CAN

FIG. 5. Partial chymotryptic digestion of 3sS-labeled precur- sor forms synthesized in the presence or in the absence of canavanine. Partial digestion of radioactive proteins with 100 pg of chymotrypsin was performed directly on gel pieces obtained from a two-dimensional slab gel. Labeled digestion products were separated on a 10 to 20rf acrylamide gradient gel and visualized by autoradiog- raphy. CONT, control; CAN, canavanine.

analyzed as described above. Recoveries of radioactive peptides on high voltage electrophoresis maps were always greater than 705.

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836 Inhibition of Pro-opiomelanocortin Maturation by Canavanine

A 3 I3 3

POMC *~ 18 4 2 5 7 7

U - M S H &END N - T E R M I N A L + t *

C H A S E

3

C A N A V A N I N E

POMC A

- MIGRATION

FIG. 6. Pulse-chase experiments on forms of POMC contain- ing or not containing canavanine. Neurointermediate lobes which had previously been preincubated in the presence or in the absence of canavanine for 16 h were then pulse-incubated for 30 min in RPMI medium containing ['Hlphenylalanine (104 Ci/mmol, 1 mCi/ml). One series of lobes was then immediately processed for electrophoresis as explained previously (26); the other series was submitted to an addi- tional chase period of 2 h in RPMI medium containing 2 mM phen- ylalanine. The chase samples were then processed as the pulse sam- ples and all were analyzed by electrophoresis on a 10% polyacrylamide slab gel containing 8.4 M urea and 0.1% NaDodSO+ Identification of POMC and some of its maturation products was done as already published (26). Absorbance tracings of fluorographs were recorded with a Joyce Microdensitometer. The position of I4C-labeled proteins (from Bethesda Research Laboratories) is also shown as a reference molecular weight scale. ,B-END, ,&endorphin.

MIGRATION ___)

:i IO 20 30 40 50

= 34,000-38,000 and corresponded to variant forms of POMC (23, 26). During subsequent chase incubations, these various forms of the precursor were progressively cleaved to yield, at the end of a 2-h chase incubation, various peptides which were identified as a-MSH, /3-endorphin, and variant forms of the NH2-terminal peptide (26). Such an experiment was repeated here for the purpose of comparison (Fig. 6A) . In order to study the maturation mechanism of precursor forms where arginine residues have been replaced by canavanine, the same experi- ment was performed on neurointermediate lobes which had been preincubated for 16 h in the presence of 10 mM canavan- ine. Fig. 6B shows that after a 30-min pulse, canavanine- containing POMC forms migrate, on the type of one-dimen- sional gel used here, as a broad band with a molecular weight slightly higher than normal POMC molecules. After a 2-hour chase incubation following the initial 30-min pulse, most of the radioactivity associated with the initial precursor band remained as high moelcular weight proteins and no radioac- tivity could be detected at the position of P-endorphin, a- MSH, or the NH2-terminal fragments. Instead, an atypical band accounting for most of the radioactivity which had disappeared from the precursor region was found with an apparent M, = 22,000-23,000. These results conclusively show that most of the precursor forms synthesized in the presence of canavanine escaped the normal maturation process ob- served in control nontreated cells. Moreover, for the small proportion of molecules which had undergone cleavage (about 25% as estimated by planimetry of the area under the peaks), the final products were of a large molecular weight, atypical of the normal end products observed in control cells. By contrast, 83% of the normal precursor forms had disappeared during chase incubations. The percentage of inhibition of prohromone conversion during incubations in the presence of canavanine can then be calculated by using the equation

Yi products with analog % products without analog

In this case it is approximately equal to 70%. I n Vitro Digestion of POMC with Trypsin-Since it has

already been postulated that some prohormones such as pro- parathyroid (34) might be readily converted to mature forms by the action of a trypsin-like enzyme alone, the next experi- ments were designed to compare the action of trypsin on

POMC MIGRATION

1 -

0 10 20 30 40 50 FRACTION NUMBER

FIG. 7. Trypsin digestion of POMC forms containing or not containing canavanine residues. POMC forms containing or not containing canavanine residues were electroeluted from two-di- mensional gels and incubated with tryp- sin (final enzyme-to-substrate ratio, 1/ 100) at 37 "C at a pH 8.0 for 0 min ( A and D ) , 30 min ( B and E ) or 60 min (C and F). Trypsin digestion was stopped by lyophilization and the samples were prepared for electrophoresis in 10% acrylamide/NaDodSOs rod gels. After electrophoresis, the gels were cut into 2- mm slices and the radioactivity of each fraction was counted A, B, and C, cana- vanine-containing POMC forms; D, E , and F, nonanalog-containing POMC forms. BPB, bromphenol blue.

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Inhibition of Pro-opiomelanocortin Maturation by Canauanine 837

isolated forms of POMC which contained or which did not contain canavanine residues instead of arginine. Metabolically labeled POMC molecules isolated by electroelution from two- dimensional gels were dissolved together with carrier BSA in 0.1 M NH4HC03 buffer, pH 8.0, and trypsin was added to a final enzyme-to-substrate ratio of 1/100 (computed on the basis of the BSA content of the sample). Control samples contained no trypsin. All were incubated at 37 "C for 0, 30, and 60 min after which incubation was stopped and the radioactive proteins were analyzed by electrophoresis on poly- acrylamide gels in the presence of NaDodS04. Control sam- ples containing no trypsin showed the same profie as the corresponding samples containing trypsin but analyzed di- rectly without having been incubated at 37 "C (not shown). This observation proved the absence of endogenous or con- taminating protease activity in the samples. Observation of the electrophoresis patterns shown in Fig. 7 indicates that, after an incubation of 60 min, both types of POMC forms (containing or not containing canavanine) had been com- pletely digested by trypsin. However, as expected, the mean size of the fragments was slightly larger for canavanine-con- taining molecules than for the control samples. After a 30-min incubation, a small peak of canavanine containing POMC forms was still observed while all the radioactivity associated with the precursor in the control sample had completely disappeared. This last observation indicates that the rate of trypsin digestion of the canavanine-containing precursor forms was somewhat slower than that of normal forms ob- tained during control experiments. These results are never- theless in sharp contrast with those of the pulse-chase exper- iments where 70% inhibition of the maturation process was observed for canavanine precursor forms.

DISCUSSION

The present study was undertaken to determine whether canavanine, an analog of arginine, could be incorporated into intermediate lobe proteins and whether, if incorporation oc- curred, maturation of POMC was affected. In order to answer the first question, we designed several experiments which allowed us to obtain proof of the incorporation of canavanine into intermediate lobe proteins through the characterization of POMC forms synthesized in canavanine-treated pituitary explants. Moreover, careful examination of our data led us to believe that substitution by canavanine occurred at most, if not all, of the arginine positions. This assertion is based on the following experimental facts: after high voltage paper analysis of the tryptic peptides from canavanine-containing POMC forms (peptides I' and 11', Fig. 3B), no fragments were found at the position where the normal NHz-terminal tryptic peptides migrated during the analysis of control samples. As explained under "Results," failure to cleave after arginine 57 (the sequence of Drouin and Goodman) (19) will result in the production of atypical large peptides which probably stay at the origin during high voltage paper electrophoresis; if only partial replacement of arginine residues had occurred, and if we assume that form I' represents a mixture of forms I and I1 where all the arginine residues have been replaced (see argu- ment 1 above), then we would have observed the synthesis of POMC forms with PI values intermediate between the PI of normal precursor forms (8.2) and that of form I' (6.0). By contrast Fig. 3B shows that there is virtually no peptide in this PI range.

The above conclusion seems to be in disagreement with the data obtained during [3H]arginine incorporation experiments, where only a net 35% inhibition over the nonspecific decrease of the overall rate of protein synthesis was observed. One has to consider, however, that under incubation conditions such

as those used in Fig. 2, exogenous canavanine concentration was about 300 times that of arginine (as ["Hlarginine). Since amino acid analogs have already been shown to compete about 1/100th as effectively as their normal counterparts for protein synthesis (34,35), it is possible that when inhibition of [3H]arginine incorporation was assessed, the canavanine con- centration was about as effective as a 3-fold dilution of [3H] arginine by nonlabeled arginine.

In the absence of exogenous arginine, it is therefore quite possible that a 16-h preincubation with 10 mM canavanine is sufficient to completely prevent incorporation of arginine res- idues into proteins. The results from Fig. 2 must therefore be considered retrospectively merely as a proof that canavanine- treated neurointermediate lobes are still capable of protein synthesis at a constant rate for at least 6 h, although half as efficiently as the control lobes.

During the analysis of pulse-chase incubation products on one-dimensional gels, POMC could be identified on the basis of its apparent molecular weight by comparison with the patterns obtained on two-dimensional gels where normal and canavanine-containing POMC forms had previously been clearly characterized. Maturation of normal POMC molecules into typical intermediate lobe end products was monitored by observing the transformation of large molecular weight pre- cursor molecules into small peptides previously identified as a-MSH, P-endorphin, and variant forms of the NHz-terminal peptide (26). It was estimated that after a 2-h chase incuba- tion, about 83% of the radioactivity associated originally with the normal precursor had been transformed into smailer pep- tides. By contrast, only 25% of the canavanine-containing POMC molecules had undergone the maturation process. The small fraction of canavanine-containing POMC molecules which was processed yielded atypical high molecular weight peptides. The failure of canavanine-containing POMC mole- cules to undergo a normal maturation process provides inter- esting clues as to the nature of the processing enzyme itself: 1) this enzyme presumably recognizes basic amino acid dou- blets only and is not capable of cleaving the precursor at the numerous lysine positions which remained unchanged after canavanine incorporation. Moreover, we have checked that trypsin, at a concentration which had been shown to selec- tively cleave the additional hexapeptide sequence of propar- athyroid hormone (36), is still capable of degrading canavanine containing POMC molecules in uitro. 2) There are two Lys- Lys sequences in the rat precursor sequence (19). One of them is situated at positions 112-113 and divides the precursor molecule into two fragments of approximately the same length. The other one is located at the carboxyl terminus of the P-endorphin sequence (positions 206 and 207). If blockage of canavanine-containing doublets had redirected the matu- ration enzyme toward the Lys-Lys sequences, one should have observed the production of atypical peptides with M , 12,000-16,000 (taking glycosylation into account). Since the abnormal peptides resulting from partial maturation of pre- cursor molecules had much higher molecular weights (around 22,000 to 23,000), it is unlikely that cleavage of the middle Lys-Lys sequence had occurred. This could mean that either the maturation enzyme recognizes only Arg-Arg or Lys-Arg doublets or that the Lys-Lys sequence is not accessible be- cause of some conformational feature of the precursor. It is more likely that production of small amounts of the 22,000- 23,000-dalton peptides during the chase period results from the slow cleavage rate of some lysine-canavanine sequences. The Lys-Arg sequence situated between ACTH and P-LPH is the first to be cleaved during the normal maturation process (26). Cleavage at this position releases /?-LPH and glycosyl- ated peptides which contain both the NHn-terminal part of

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838 Inhibition of Pro-opiomelanoco

the precursor and the ACTH sequence and whose molecular weight (23,000 to 25,000) is very close to that of the canavan- ine-containing cleavage products. Production of these 22,000- to 23,000-dalton peptides could therefore occur through the slow hydrolysis of the precursor between the ACTH and the P-LPH sequences.

In conclusion, the incorporation of canavanine into POMC molecules has clearly been proven to considerably slow down prohormone conversion. This in turn could be used to promote precursor accumulation into isolated neurointermediate lobes so as to prepare large quantities of the protein for structural analysis.

Acknowledgments-We thank Dr. M. Mamet for critical reading of the manuscript and S. Kieran and M. Smit for their help in preparation of the manuscript and figures. Synthetic peptides used in these studies were generous gifts from Dr. M. Chretien.

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P Crine and E Lemieuxadrenocorticotropin and beta-lipotropin.

maturation of pro-opiomelanocortin, the common precursor to Incorporation of canavanine into rat pars intermedia proteins inhibits the

1982, 257:832-838.J. Biol. Chem. 

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