Studies on the Conversion of Proinsulin to Insulin FOR A CHYMOTRYPSIK-LIKE CLEAVAGE IN THE CONKECTING PEPTIDE REGION OF IKSULIn’ PRECURSORS IN THE RAT*

Abstract A chymotryptic-like fragment of the rat proinsulin connecting peptide (C-peptide) has been isolated from whole rat pancreas. Amino acid analysis and partial sequence determination showed the fragment to contain the NH2-terminal 22 residues of C-peptide I. A radioactive fragment of similar electrophoretic mobility was also isolated from rat pancreatic islets which had been incubated with [3H]-leucine. Stepwise Edman degradation of the radioactive fragment showed the positions of its leucine residues to be identical with those of the unlabeled material. Homogenization of [3H]leucine-labeled rat C-peptide with whole pancreas and extraction under conditions used for the isolation of the fragment did not result in cleavage of the C-peptide to the fragment. Furthermore, the fractional ratio of fragment to C-peptide (normally about 0.2) did not increase when rat islets were incubated with [3H]leucine for 1 hour and then incubated with unlabeled leucine for 1, 2, or 3 hours. Treatment of a mixture of [3H]leucine-labeled proinsulin and proinsulin intermediates with high amounts of trypsin resulted in the release of the C-peptide fragment as well as desglutamine (residue 31) C-peptide. It is concluded that a chymotrypsin-like cleavage in the C-peptide region of proinsulin or in one of its intermediates occurs normally during the conversion of proinsulin to insulin in the rat.

GOBS?' SUMMARY A chymotryptic-like fragment of the rat proinsulin connecting peptide (C-peptide) has been isolated from whole rat pancreas.
Amino acid analysis and partial sequence determination showed the fragment to contain the NH2terminal 22 residues of C-peptide I. A radioactive fragment of similar electrophoretic mobility was also isolated from rat pancreatic islets which had been incubated with [3H]leucine.
Stepwise Edman degradation of the radioactive fragment showed the positions of its leucine residues to be identical with those of the unlabeled material.
Homogenization of [3H]leucine-labeled rat C-peptide with whole pancreas and extraction under conditions used for the isolation of the fragment did not result in cleavage of the C-peptide to the fragment.
Furthermore, the fractional ratio of fragment to C-peptide (normally about 0.2) did not increase when rat islets were incubated with [3H]leucine for 1 hour and then incubated with unlabeled leucine for 1,2, or 3 hours.
Treatment of a mixture of [3H]leucine-labeled proinsulin and proinsulin intermediates with high amounts of trypsin resulted in the release of the C-peptide fragment as well as desglutamine (residue 31) C-peptide. It is concluded that a chymotrypsin-like cleavage in the C-peptide region of proinsulin or in one of its intermediates occurs normally during the conversion of proinsulin to insulin in the rat.
The biosynthesis of irlsulin occurs by way of the single chain precursor proinsulin (1). l'hc conversion of proinsulin to its hormone product in the secretory granules of pancreatic P-cells (1,2) is not yet fully understood, although chemical and biosynthetic studies have indicated a likely course to this proc'ess. Steiner et al. (3) first noted that the conversion of proinsulin to an insulin-like substance (desalanine insulin) proceeded readily during the digestion of the radioactively-labeled precursor with trypsin.
Although Grant and Coombs (4) pointed out that a trypsin-like enzyme may well serve to convert proinsulin to insulin in many fishes, enzymes having only trgptic specificity will not account for all aspects of the conversion of proinsulin to insulin in higher animals.
The determination of the primary structures of bovine (5) and porcine (6) proinsulins suggested that enzymes with trypsill-like and carbosypeptidase Is-like activities would be required for the conversion of proinsulin to intact insulin.
Kemmler et al. (7), in fact, showed that bovine proinsulin is readily and quantitatively converted to intact insulin and C-peptide' when the precursor is treated with trypsin and carboxypeptidasc I< under conditions where the tryptic activity is rate-limiting.
h partially cleaved form of porcine proinsulin corresponding to the first of the above structures has also been isolated (9). Evidence accumulated from studies of the incorporation of radioactive amino acids into proinsulin and from the conversion of the precursor into insulin in several in vitro preparations of islet tissue (10-12) has indicated the likelihood that these structures represent intermediates in the convrrsion of proinsulin to insulin.
A second type of proinsulin intermediate showing a chymotrypsin-like cleavage in the C-peptide region of the molecule has been isolated from commercial preparations of crystalline porcine insulin (9). The existence of such an intermediate should result in the appearance of a chymotryptic-like fragment of the C-peptide as well as the intact C-peptide among the final products of the conversion reactions.
Studies of proinsulin conversion in the rat, especially with regard to the condition of the C-peptide, provide an excellent probe for such a chymotrypsin-like cleavage. Since the two C-peptides found in the rat contain penultimate arginine residues (13,14), cleavage at any of several possible sites by a chymotrypsin-like enzyme would give rise to more acidic peptides.
These pcptides should be easily separated from the undegraded material because of their charge difference at low pH.
The existence of fragment of the rat C-peptide was suggested by Clark (15) during his stud& of the biosynthesis of insulin in isolated pancreatic islets and his studies of the isolation of C-peptide from whole pancreas.
WC report here the identification of an iYH8-terminal fragment of the rat C-peptide isolated from whole rat pancreas.
Control studies have indicated that the fragment is not an artifact of isolation.
Kosynthetic evidence is also presented suggcstiug that the fragment arises within the pancreatic P-cell from a proinsulin intermediate which has undergone a chgmotrypsiii-like cleavage in its C-peptidc region.
MI'THODS 1 , isolation 0.j" Unlabeled C-peptide Fragmen--The isolation of the C-peptide fragment (peptidc A) was carried out as previously described for the isolation of the intact C-peptide (14). Au ether-ethanol-insoluble protein fraction was isolated from au acid-ethanol extract of whole pancreas and the resulting material was gel-filtered in 3 M acetic acid on Ko-Gel I'-30 (14). Paper electrophoresis in 300/, formic acid of the protein peak containing insulin resulted in the isolation of a fraction containing the fragment as well as thr two C-pcptides.
Thr fragment migrated less rapidly on paper than the intact C-peptide, but complete separation was not possible on the preparative scale.
'I'hc mixture of peptidc and fragment was applied to a QAE-Sephades column in 0.05 RI pyridinc formate buffer at pH 5.0 alld the pcptide material clutcd with a stepped pH gradient as prel-iously described (I 4). i1fter the $1 of the column effluent had decrtascd to about 2.5, the column n-as eluted Tvith 507; acetic acid to remove cvcu the most acidic peptide material. The C-l)el)tide fragment elutcd with the acetic acid front and was detected by spotting aliquots of each fraction on paper followed by visualization with Iliuhydrin spray. The fragment was then further purified by ~)npcr clectrophoresis in 10% pyridille brought to $1 6.5 with a(+ctica acid. The yirld of material from 400 g of pancreas was about 0.5 mg. [solution and fncubation of Rat Islets-The rats uwd in these studies were 400 to 500 g malrs of the Sprague-Dawley strain, which were injected with c~ortisolle-a(~etate (10 mg per kg body weight every 211d da)-) for 5 to 15 days l)rior to sacrifice in order to obtain higher yields of pallc~rc~atic islets (3). Treatment with cortisone-acetate causes some hypc~rplasia of the islets (16) and seems to enhallcc their insulin biosynthetic: activity without altering the over-all kinetics of proinsulin processing (3). Pancrcatic islets were isolatcxd from millrc>d tissue essentially as dcscribed elsewhere (17). Crude collagcnase TT-as obtained from the \\'orthington l~iocl~emi~al Company.
Hanks' salt solution and the incubation medium were prepared fresh and adjusted to pII 7.4 by gassing with 95$', 02-5% COZ. The tracer used ill all experiments was I,-14, 5-311]lcucinc (Amersham Radiochemical Center) having a specific: activity of 46.9 Ci per mmole. L)urillg each esperimellt, 75 to 600 islets were incubated in 150 to 300 ~1 of medium colltailling 0.1 PCi per ~1 of [3H]leucine. When the incubation medium was to be analyzed, extreme care was taken to obtain islets free from contamination by acinar tissue. After incubation at 37" under the appropriate condi-tions, the islets and sometimes the incubation medium were extracted in acid-ethanol by a modification of the Davoren procedure (I@, using 1 mg of insulin or proinsulin and 0.1 mg of leucinc as carriers. Islet extracts were gel-filtered on a column (1 x 50 cm) of Bio-Gel P-30 equilibrated with 3 M acetic acid, and 1.5 ml fractions were collected.
Ultraviolet absorbance of the carrier protein was measured at 275 nm and aliquots from each fraction were counted in 15 ml of Triton-toluene scintillation fluid (17) ill a Packard model 527 liquid scintillation spectrometer. Various fractions were then pooled and brought to dryness by a rotary evaporator.
Insulin-and C-peptide-containing fractions were dissolved in 3Oc-/0 formic acid and were subjected to paper electrophoresis in that solvent for 5 hours at 7.5 volts per cm.
Isolation of [311]Leucine-labeled C-peptide and C-peptide Fragment-After electrophoresis in 3Ooi, formic acid of the material isolated from the incubation of rat islets with [FI]leucine and after localization of the radioactive peptides on the electrophorctograms (see later), the appropriate bands were cut from the paper and the peptides eluted using 500/b acetic acid. The acetic acid was then removed by rotary evaporation.
In one control experiment, the radioactive intact C-peptide was homogenized T&h 1 g of whole rat pancreas in acid-ethanol and the homogenate further treated as for the isolation of the unlabeled material (I 4). These isolation steps included precipitation of contaminating proteins at pH 8.5, precipitation of insulin and C-peptide by the addition of absolute ethanol and ether at pH 5.3, gel filtration in 3 M acetic acid, and electrophoresis in 30% formic acid.
A scparatc control digestion of 100 pg of a mixture of the C-peptide fragmeut, the (I-pcptide, and the rat insulitis with 20 pg of trypsin was carried out concurrently. Localization and Quantitation of 311-babeled Peptide Components on Paper-Whell large amounts of radioactive material ( > 15,000 dpm) WIT prcwnt, for esamplc wlw11 islets were incubated with [W]leu~iuo for 6 hours, the paper c~lcctrophoretograms were scanned on a Vanguard model 880 Chromatogram Scanner at a sl)clc:d of 6 illchcs per hour. MThen lesser amounts of radioactivity were prcscnt, we obtained improxwl wsults by counting paper striljs ill the liquid scintillation spwtrometer. The clectrophoretogram was cut iu 2-mm strips pc~rpelldicular to the directioll of migration and each strip) was caountcd for radioactivity in 10 ml of Triton-toluene scintillation fluid. The recovery of radioactivity was approsimatcly 30 $;,. Other Procedures-Amino acid analysis was performed on a model 12OC or 121 Beckman amino acaid allalyzcr using standard methods.
Stepwise Edman degradation n-a.s performed by the semi-micro procedure recently dcwribcd by this laboratory (19). l'hcnvlthiollydalltoill derivativc>s of the amino acid residues were identified by thin layer chromatogral)hy 011 silica gel sheets (20). Ikriug the degradation of the j311]leuc~ilie-labeled peptide, aliquots of the phenylthiocarbamyl amino acids were counted for radioactivity in 10 ml of Tritoll-tolucne scintillation lluid.
-1ftrr incubation for 0, 3.3, 10, 20, or 60 min at 21", or for 60 min at 21" followed by 60 min at 37", the reaction was stopped by the addition of glacial acetic acid and the samples were applied to the long column of the amino acid analyzer for the determination of free amino acids released during the enzyme digestion. RESULTs: Suitable methods for the isolation of insulin and C-peptide from either whole pancreas or isolated pancreatic islets are now well established (8,10,14,18,19). The purification procedure used here, which included acid-ethanol extraction, ethanol-ether precipitation, and gel-filtration, results in a highly purified mixture of C-peptide and insulin. Fig. 1 shows the pattern of radioactivity obtained when the insulin-and C-peptide-containing fraction resulting from the incubation of rat pancreatic islets with [3H]leucine is submitted to paper electrophoresis in 30% formic acid. The sizable amount of radioactivity occurring in a peptide migrating to a position slightly nearer the origin than the C-peptide led us to suppose that this peptide might be significant in normal p-cell metabolism.
A peptide with identical electrophoretic mobility also appears in the insulin-and C-peptide-containing fraction isolated from whole rat pancreas (see legend Fig. I). Each of the two rat C-peptides maintains a net charge of +2 in 30"/, formic acid, pH 0.9, and both peptides migrate to the same position after paper electrophoresis (14). At that pII, rat insulins 1 and 2 maintain net charges of +7 and +6 (al), respectively, resulting in their partial separation as shown in Fig. 1.
The amino acid composition of the slower migrating peptide A obtained from whole rat pancreas is presented in Table I. The peptide not.ably lacks arginine, lysine, histidine, isoleucine, phenylalanine, and tyrosine.
Furthermore, the composition of peptide A corresponds rather well to that expected from the NH2- Preliminary purification of the fraction from 600 islets incubated with [aH]leucine for 6 hours included ethanol-ether precipitation at pH 5.3 and gel-filtration on Bio-Gel P-30, as described under "Methods." The positions and appearance of ninhydrin-positive substances purified from whole rat pancreas using identical procedures are shown in the lower part of the figure. Peptide A represents the putative C-peptide-like material; peptide C, the C-peptide; peptides I-l and I-2, insulins 1 and 2, respectively. Radioactivity was measured on a Vanguard strip scanner. The direction of migration is from anode to cathode. terminal 22 residues of rat C-peptide I (Table I), notwithstanding the relatively high alanine coiltent which may arise from a minor contaminant.
Kc purified the [3H]leucine-labeled peptides by paper electrophoresis in 30% formic acid in order to avoid possible loss of the very small amount of material due to nonspecific binding to QAE-Sephades in dilute, aqueous buffers. Fig. 2 (left) shows the electrophoretic patterns obtained separately from the V-labeled C-peptide and the 311-labeled peptide A after the earlier purification of the peptides by paper electrophoresis.
We were able to obtain peptide A in a purity of about 97%. The labeled, principal C-peptide fraction was contaminated with approximately 12% peptide A, but was considered to be of sufficient purity to be used in a probe for possible artifacts arising from the isolation procedures.
A portion of the Wlabeled C-peptide was homogenized with 1 g of rat pancreas and the homogenate treated to obtain the fraction containing peptide A, C-peptide, and insulin. Electrophoresis in 30% formic acid revealed that the reisolated 3H-labcled C-peptide-like material contained 13 '$0 peptide A (Fig. 2, right) indicating that peptide A did not arise from the degradation of the C-peptide during isolation.
The [3H]leucine-labeled peptide A and the unlabeled peptide A were separately subjected to 15 cycles of Edman degradation, with the results shown in Fig. 3. The partial sequence of the unlabeled material is identical to that of the NHz-terminal 15 residues of intact rat C-peptidc I. Furthermore, the positions of the leucine residues in the labeled material are identical to those in peptide A obtained from whole pancreas.
Treatment of unlabeled peptide A with carboxypeptidase A revealed the COOH-terminal sequence of the peptide to be Leu-Gin-(Thr) ( Table II).
Since the approximately 0.5 mole of threonine present in 1 mole of peptide A (Table I) appeared early in the carboxypeptidase A digest, it may well occur as the COOH terminus in a portion of the peptide A molecules.
It is presumed that free amino acids other than leucine, glutamine, and threonine were not detected in the carboxypeptidase A digest because the Gly-Asp sequence preceding Leu-Gln in the rat C-peptides blocks against further carboxypeptidase action (14). Fig. 4 shows the results of a pulse-chase experiment designed Amino acid composition of the NH%-terminal fragment of the rat C-peptide All results are expressed as moles of amino acid per mole of peptide.
Hydrolysis proceeded in 6 N HCI at 110" for 20 hours. For comparison, the amino acid compositions of the NH?-terminal fragments (residues 1 to 22) of the two rat C-peptides are also presented.
Amino acid residues not reported below are absent from the peptides lmder consideration.  to determine whether or not the proportion of peptide A to Cpeptide increased as proinsulin synthesized during a l-hour pulse with [3H]leucine was converted to C-peptide plus insulin.
Also, the ratio of peptide A to C-peptide did not increase when pancreatic islets incubated in the presence of [%leucine with high amounts of glucose for 3 hours were then further incubated with low amounts of glucose for another 4 hours. 2 We also considered the possibility that peptide A might arise biosynthetically from the proinsulin and proinsulin intermediate fraction present in P-cells of the rat pancreatic islets. The digestion of bovine proinsulin with trypsin results in the liberation of C-peptide with an additional lysine residue at its COOH terminus (8). Uigestion of rat proinsulin with trypsin, however, would result in the formation of the desglutamine C-peptide since the COOH-terminal Arg-Gln sequences of the two rat C-peptides The direction of migration is from anode to cathode.
are cleaved by trypsin (13). Thus, the C-peptide-like material obtained from tryptic digestion of rat proinsulin should retain the electrophoretic mobility of the unmodified C-peptide. As shown in Fig. 5, tryptic digestion of a fraction containing proinsulin and proinsulin intermediates obtained after a g-hour incubation of rat islets with [3H]leucine resulted in the liberation of peptide A as well as C-peptide or desglutamine C-peptide or both.
The control digestion of an unlabeled mixture of C-peptide, peptide A, and insulins 1 and 2 under similar conditions showed that the migrations of peptide A and C-peptide were unchanged, although the migration of the insulin-like components was slowed considerably (cf. Fig. 1) due to cleavage carboxyl to Lys 3 and Arg 22 in the rat insulin U chains (21). We also studied the products resulting from the tryptic digestion of proinsulin obtained from rat islets incubated with [ and appearance of ninhydrin-positive substances resulting from a trypsin digestion of a mixture of peptide A, Cpeptide, and insulin obtained from rat pancreas is shown in the lower part of the figure (cf. Fig. 1). The direction of migration is from anode t,o cathode. only 20 min. This proinsulin preparation, which should contain few if any partially cleaved proinsulin intermediates (3), was cleaved entirely to C-peptide-dcsglutamine C-peptide and desoctapeptide insulin.
The lack of peptide A formation during this digestion precludes the possibility that the peptide X appearing iu Fig. 5 had arisen from nonspecific proteolgtic activity in our preparation of L-l-tosylamide-2-phcr~ylethylchloromethyl ketonetrypsin.
It is concluded, therefore, that fragment A arises as a result of the conversion of a two-chaiued proinsuliu intermediate containing a single chymotryptic-like cleavage within the COINnetting polypeptide segment. u1scuss10x The identical electrophoretic mobilities of one component of a fraction of whole rat pancreas caontaining insulin and C-peptide and of a biosynthetically-labeled component isolated from rat islets led us to question whether or not the component (peptide A) might be structurally related to the normal products arising from the conversion of insulin to proinsulin.
Amiuo acid analysis and partial sequence determination of peptide A showed that this peptide fraction consists mainly of the NH?-terminal 22 residues of the intact rat C-peptide I, the sequence of which is Glu-Val-Glu-Asp-Pro-Gln-Val-Pro-Gln-Leu-Glu-Leu-Gly-Gly-Gly-Pro-Glu-Ala-Gly-Asp-Leu-Gl~~-'l'hr-Leu-~~la-Leu-Glu-Val-Ala-Arg-Gin (14). In order to demonstrate that the biosynthetic peptide A was identical to that isolated from whole pancreas, we undertook the partial sequence determination of the labeled peptide. The results showed that the leucine residues occurred in the labeled material in the same positions as in the unlabeled material (Fig. 3), suggesting that the two peptides are, in fact, identical.
The precipitation of this fragment of the rat C-peptide from an acid-ethanol extract of pancreas by ether-ethanol is perhaps unexpected.
Although smaller and more acidic than the C-peptide, it apparently maintains many of the chemical and physical characteristics of the intact material.
The finding that intact [WIleucine-labeled rat C-peptide was not degraded to the NHt-terminal fragment during homogenization with whole pancreas and subsequent reisolation eliminates the possibility that peptide A is an artifact of isolation, arising either from residual enzymatic activity in the exocrine pancreas or from acid-catalyzed cleavage.
The stoichiometry of amino acid residues in peptide A (Table I) suggests that the process resulting in its formation is specific. In assigning a likely catalytic source for the formation of the NHz-terminal fragment, it is pertinent to note that two sites in the C-peptide in the region distal to Leu 21 are sensitive to chymotrypsin.
We previously reported that chymotrypsin cleaved the intact rat C-peptides between Gln-Thr (residues 22 and 23) (14), whereas Markussen and Sundby (13) found chymotryptic cleavage to occur between Leu-Ala (residues 24 and 25). The results of the digestion of peptide A with carboxypeptidase (Table II) suggest that the peptide arises from a chymotrypticlike cleavage at one or the other of these sites.
Thus, the early release of a small amount of leucine and the release of approximately 0.5 mole 'i: of threoniue by carboxypeptidase A may indicate heterogeneit,y ill the COOH termil;us of peptitle A. It is possible that au initial chymotryptic-like cleavage occurred between residues 24 and 25 yielding a peptitlr with the COOKterminal sequence Leu-Ghl-'1 hr-Lcu. C'arbosypcptidase activity within the P-cell might slowly degrade the matcrial to a mixture of several peptides lacking 1 or 2 neutral amino acid residues.
On the other hand, the threoniue present ill the carboxypeptidase digest may arise from a minor coutaminnnt. The unequivocal COOI-I-terminal srqucnce Lcu-Gin might then result from a chymotrypt,ic-like cleavage between residues 22 and 23. In cither case, we cannot exclude the 1:ossibility that the principal cleavage enzxrne is similar to papain, cathepsili I), or cathcpsin I), ,siuce these enzymes and a-chgmotrypsin can show overlapping substrate specificities (22)(23)(24).
Since the proportion of the NHs-terminal fragment to the Cpeptide does not increase as the C-peptide ages within the P-cell (Fig. 4), II--e can exclude the possibility that the fragment arises directly from a chymotrypsill~likc cleavage of the C-peptidc itself. Had such an enzymatic cleavage occurred, the ratio of fragment to C-peptide would have illcreased during the 1011ger periods of chase, following the reaction COUI'SP, Cpeptide + SIIn-terminal fragmcut.
The lack of increase ilr the ratio of fragment to peptide, howler, might also be explained by the esistence of the following ~equcnce of reactions: C-peptidc -+ ?;H-trrminal fragment + Fmall pcptides. If thr first reaction in this requence were ralc-limitiiig, the ratio of Nl%-terminal fragment to C-pcptide might not rise until most of the C-pcptide had been degraded.
This reaction sequence apparently does not play a major role in the formation of the fragment since the ratio of Cpeptide-like material to insulin-like matel,ial remains COIIstant under a ITide variety of incubation conditions (25). The results shown in Fig. 5 clarify the origin of the NH-tcrminal fragment of the rat C-pcptide.
Trypsiii treatment of a mixture of rat proinsulin and proinsulin iutermediates released the X&terminal fragment as well as the desglutamirrc Cmpcptide. These results suggest that in islet tissue this fragment arises from a proinsulin intermediate which has been cleaved in its C-peptide region, rather than from secondary cleavage of the free C-pcptidc. The release of the chymotryptic-like fragment from this precursor in viva may then result from the conversion of the proinsulin illtermediates to insulin.
Furthermore, the retention of the fragment within the secretory granules of the @-ccl1 results in its The major pathway for proinsulin conversion is shown in heavy lilzes. The sites on either side of the C-peptide filled by pairs of basic amino acid residlles are indicated by circles and scjusres. Appropriate cleavage at, these sites to yield instl!in and C-peptide requires both trvusin-like and carboxvpeptidase B-like activities. The slash "_ I_ _ represents a chymotrypsin-sensitive site in the C-peptide region of nroinsulin.
The NH? termini of the A and B chains of insulin ar,' indicated by dots. appearance in the incubation medium when pancreatic islets arc stimulated by high glucose concentrations to secrete insulin.2 A diagrammatic representation of the possible routes of formation of the NHS-terminal C-peptide fragment from proinsuliu iI]tcrmediates cleaved in their C-peptidc regions by a chymotrypsinlike enzyme is presented in Fig. 6. The major routes for proinsulin conversion to intact C-peptide and insulin by way of trypsin-like and carbosypeptidase U-like activities have been suggested by biosynthetic studies in islet tissue (10, ll), by enzymatic studies in vitro (7), and by the isolation of compounds analogous to Intermediates 1 and 2 from commercial crystalline preparations of insulin (5, 8,9). Two other presumptive intermediate forms have been isolated from crystals of porcine insulin (9), but not from crystals of bovine insulin (5, 8). They are porcine proinsulin split in its C-ppptide region between residues 54 and 55, and desnonapeptide porcine proinsulin (lacking residues 55 to 63) (9). These forms are analogous to Intermediates 3 and 4, respectively (Fig. 6). Chance has suggested that the split porcine proinsulin may arise by way of a chymotrypsin-like cleavage of the sequence Leu-Sla (residues 54 and 55), a site known to be sensitive to chymotrypsin (6,9). Ilesnonapeptide porciile proinsulin may arise from a precursor analogous to Intermediate 3 by way of further cleavage at the Arg-Arg sequellce at the junction with the insulin A chain, or from a precursor analogous to Intermediate 1 by way of a chymotrypsinlike activity (9).
Clark and Steiner (10) studied proposed intermediates of rat proinsulin isolated either from crystals of rat insulin or from rat pancreatic islets incubated with [3H]phenylalanine.
The intermediates were not fully characterized and may have consisted largely of components corresponding to Structures 1 and 2 in Fig. 6. Nevertheless, the data presented here indicate that proinsulin intermediates similar in structure to Intermediates 3 or 4 do occur during the normal course of proinsulin conversion in the rat. The chymotrypsill-like cleavage of proinsulin or of Interrnediates 1 and 2 is apparently less rapid than the over-all rate of proinsulin conversion to insulin.
Thus, 15 to 25% of the C-peptide-like material released during proinsulin conversion appears as the NI-Iz-terminal fragment of the C-pcptide. The extent to which each of the Intermediates 3,4, and 5 ( Fig. 6) tolltributes to the formation of this fragment is a matter for further investigation.
The NHz-terminal fragment of the C-peptide isolated from rat pancreas appeared both by amino acid analysis and by sequence determination to arise wholely from proinsulin I. Although a similar fragment corresponding to proinsulin II might have been lost during isolation, elements of structural specificity may limit the action of a chymotrypsin-like enzyme to only one of the two rat proinsulins.
Structural specificity must play a role in the chymotrypsin-like cleavage of rat proinsulin I since cleavage al'parently does not occur in the same position of the free C-peptide during the biosynthesis and storage of insulin.
Such selectivity may also explain the apparent lack of a chymotrypsin-like cleavage of proinsulin or proinsulin intermediates to J ield NHS-terminal fragments of the C-peptide containing residues 1 to 10 or residues 1 to 12 (14). T17e cannot exclude, however, the existence of small amounts of such cleaved forms.
The biological significance of the chymotrypsirl-like cleavage of rat proinsulin or proinsulin intermediates to yield a C-peptide fragment is not clear. This cleavage, which likely occurs after correct disulfide bond formation has taken place, should not illterfere with the normal conversion of proinsulin to insulin.
Athough a physiological function for the free C-peptide has not yet been found (26), its presence in the plasma (27) suggests the possibility that the peptide or some part of it may be biologically functional.
It is also possible that the chymotrypsin-like cleavage represents the degradation of a peptide which is no longer useful. In view of the possible evolutionary derivation of the proinsulin converting enzymes from the esocrine pancreatic proteases (3)) it is not surprising that low levels of chymotryptic-like activity may occur in ,&granules.
However, the presence of chymotrypsin-like and trypsin-like enzymes in the secretory granules of mast cells (28) suggest that such enzyme activities may be widely distributed throughout the organism and that they may serve a variety of purposes.
As mentioned previously, our studies 011 the conversion of proinsulin to insulin in the rat were aided by the charge difference between the intact C-peptide and its NHP-terminal fragment. The isolation of requisite intermediates for a similar chymotrypsin-like cleavage in porcine proinsulin (9) suggests that the occurrence of such activities in the conversion of proinsulin to insulin is not restricted to one species. The apparent lack of such intermediates in the case of bovine proinsulin may simply relate to the lack of a site sufficiently sensitive to chymotrypsin in the C-peptide region of that molecule (5, 8).
It may be anticipated, however, t.hat fragments of the C-peptide will be found in extracts of pancsrcas from other species.