THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 246, No. 5. Issue of March 10, pp. 1365-1374, 1971

Printed in U.S.A.

Isolation and Characterization of Proinsulin C-Peptide from Bovine Pancreas*

(Received for publication, September 8, 1970)

DONALD F. STEINER,~ SOOJA CHO, PHILIP E. OYER,§ SUSAN TERRE, JAMES D. PETERSON, AND ARTHUR H. RUBENSTEIN

From the University of Chicago, Departments of Biochemistry and Medicine, Chicago, Illinois 60637

SUMMARY

Although the enzymes that catalyze the transformation of proinsulin to insulin have not yet been identified, studies of intermediate forms of proinsulin isolated from bovine pan- creas indicate the existence of a mechanism in which the interchain connecting peptide is cleaved with elimination of a pair of basic residues from each end to yield insulin and the intact remainder of the connecting peptide segment. This acidic peptide, designated the C-peptide, has been isolated from fresh bovine pancreas and purified by means of a procedure involving acid-ethanol extraction, gel filtra- tion, carboxgmethylcellulose chromatography, paper electro- phoresis, and partition chromatography. ‘The peptide was found to occur on an equimolar basis with insulin, as expected from the stoichiometry of the cleavage process. The yield of both peptides was approximately 10 nmoles per kg of pancreas. Analysis of the amino acid sequence of the pep- tide and its chymotryptic fragments demonstrated its structural identity with the authentic proinsulin C-peptide prepared by tryptic digestion of the bovine proinsulin inter- mediate fraction. These findings are in accord with the hypothesis that the cleavage of proinsulin occurs within newly formed secretory granules and the products are retained therein until they are discharged together by means of fusion of the granule membrane with the cell membrane in the process of emiocytosis.

It is now well established that proinsulin, a single polypeptide chain protein, is the biosynthetic precursor of insulin (l-5). The prohormone has been isolated from crystalline preparations of bovine (6, 7), porcine (8), human (9), and rat (10) insulin, where it occurs in small amounts ranging from 0.1 to 2% of the insulin, and the primary structures of the porcine (8) and bovine (11) proinsulins have been elucidated. The single

* This work was supported by United States Public Health Service Grants AM-04931, AM-13914, and AM-13941.

$ Recipient of a United States Public Health Service Research Career Development Award.

4 Supported by Public Health Service Training Grant HD-00001 from the National Institute of Child Health and Human Develop- ment.

polypeptide chain of both bovine and porcine proinsulin is ordered as: NH%-B chain .Arg .Arg . C-peptide . Lys . Arg . A chain. COOH. The C-peptide portion of the connector polypeptide contains acidic ’ residues, proliie, glycine, alanine, valine, and leucine, but has no aromatic residues, histidine, or basic amino acids. The sequence of the 26-residue bovine C-peptide is: Glu-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu-Leu-Ala- Gly-Gly-Pro-Gly-Ala-Gly-Gly-Leu-Glu-Gly-Pro-Pro-Gln.

Studies of the minor components of crystalline bovine insulin have revealed the presence of several partially cleaved forms of proinsulin, which appear to be intermediates in the conversion of proinsulin to insulin (6, 12, 13). The most abundant of these are two closely related forms that have undergone cleavage to liberate the carboxyl end of the B chain with loss of the Arg- Arg dipeptide (Residues 31 and 32), or alternatively, to liberate the amino terminus of the A chain with loss of the Lys-Arg dipeptide (Residues 59 and 60), as shown in Fig. 1 (12). Because of their nearly identical structure and net charge over a wide pH range, these two forms are indistinguishable in many electro- phoretic and chromatographic systems. Together they com- prise about twice as much of the crystalline protein as intact proinsulin, and are far more abundant than a variety of other related products which occur in trace amounts and are possibly produced by autolysis preceding or during isolation.

Although the proteolytic system responsible for cleaving proinsulin has not yet been isolated, consideration of the struc- tures of the intermediate forms suggests that native insulin and the intact C-peptide, as well as both pairs of basic amino acids (either as single residues or dipeptides), are liberated during the reaction (Fig. 2). Evidence from biological and immunological studies (10, 12-14) supports the existence of such a cleavage mechanism within the p-cells of the pancreas and indicates that the C-peptide and insulin are both packaged in secretory granules and subsequently liberated together into the circulation (15, 16). It was thus anticipated that the C- peptide should occur in normal pancreatic tissue on an equimolar basis with insulin, and this possibility was confirmed in pre- liminary experiments (17). The purpose of this report is to describe in detail the isolation of C-peptide from bovine pan- creas and to present evidence that the pancreatic peptide is identical in structure with the proinsulin C-peptide isolated from tryptic digests of the bovine proinsulin intermediate fraction. A correction in the amino acid sequence of the bovine proinsulin connecting peptide (12) is also reported.

1365

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1366 Bovine Proinsulin C-Peptide Vol. 246, No. 5

I (60-70%)

FIG. 1. Diagrammatic representation of the two major partially cleaved intermediate forms of bovine proinsulin. Numbers repre- sent distance in residues from the amino-terminal phenylalanine of the B chain.

FIG. 2. Diagrammatic representation of the proposed major products of the in vivo conversion of bovine or porcine proinsulin to insulin.

MATERIALS AND METHODS

First crystals of bovine insulin and the crude proinsulin fraction, separated by gel filtration of insulin, were kindly supplied by The Novo Company (Copenhagen). Fresh bovine pancreas was obtained from The Illinois Packing Company (Chicago). Tosylamido-2-phenylethyl chloromethyl ketone- treated trypsin was prepared by treating Novo Trypure crystal- line bovine trypsin with n-l-tosylamido-2-phenylethyl chloro- methyl ketone according to the procedure of Wang and Carpenter (18). ar-Chymotrypsin and carboxypeptidase A (diiso- propyl fluorophosphate-treated) were supplied by Worthington. Standard phenylthiohydantoin derivatives of amino acids were obtained from Sigma and from Mann. Dansyl’ amino acid standards were prepared from amino acids supplied by Cal- biochem. Dansyl chloride and Sequanol grades of phenyliso- thiocyanate reagent, anhydrous trifluoroacetic acid, pyridine, and triethylamine were obtained from The Pierce Chemical Company, Rockford, Illinois. Thin layer cellulose (Catalogue Number 6064) and silica gel (Catalogue Number 6060) chromato-

1 The abbreviations used are: dansyl, dimethylaminonaphtha- lened-sulfonyl; Cm-, carboxymethyl-.

graphic sheets were from Eastman. Polyamide thin layer sheets were supplied by The Cheng-Chin Trading Company (Gallard Schlesinger Corporation, New York). Formylated cellulose powder (Cellex XF-1) and Bio-Gel P-30 were obtained from Bio-Rad (Richmond, California). Ion exchange celluloses (DE-23 and CM-23) and No. 3MM chromatographic grade paper sheets were obtained from Whatman. Antisera against bovine proinsulin was prepared in guinea pigs as described else- where (19). Rabbit anti-guinea pig globulin serum was pur- chased from The Sylvana Chemical Company, Orange, New Jersey.

Isolation of Bovine Proinsulin Intermediate Fraction--A crude proinsulin fraction was separated from first crystals of bovine insulin by gel filtration on columns of Sephadex G-50 (6). Highly purified (>98% by polyacrylamide gel electrophoresis) intermediate fraction was separated by gradient elution chroma- tography on carboxymethyl (CM-23)- and diethylaminoethyl (DE-23)-cellulose columns in urea-containing buffers as de- scribed elsewhere (6, 12). The protein was desalted in 1 M

acetic acid over Sephadex G-25 and lyophilized (NBlD-E). Tryptic Digestion of Intermediate Fraction-About 60% of the

intermediate fraction (Form I, Fig. 1) consists of material lack- ing the Lys-Arg dipeptide (Psg-Pm). A single tryptic split in this molecule at P32-P33 (Arg-Glu) would be expected to liberate the C-peptide (P~~-Pss), whereas tryptic digestion of the remain- ing 40% of the intermediate fraction would liberate similar pep- tides having additional COOH-terminal lysine or Lys-Arg. In preliminary experiments, paper electrophoresis in 30% formic acid of the tryptic cleavage products arising from the interme- diate fraction (1% trypsin (w/w), pH 8.2) confirmed the rapid liberation in 2 to 3 min of both the C-peptide (PZ3-P& and the dibasic peptide (P33-P60), which was then slowly transformed to monobasic peptide (P83-P59). These peptides were identified on paper electrophoretograms by means of their relative mobilities, their positive reaction with ninhydrin (Ninspray, Sigma), their lack of staining with the Pauly reagent, and their reaction with the Sakaguchi reagent when arginine was present.

Based on the foregoing observations, 170 mg of intermediate fraction (NBID-E) were incubated with 5 mg of tosylamido-2- phenylethyl chloromethyl ketone-trypsin in 17 ml of 0.2 nf Tris-HCl buffer at pH 8.2 for 15 min at 37”. The mixture was then acidified with 1 ml of glacial acetic acid and desalted on a Sephadex G-25 column eluted with 0.1 M acetic acid. The protein-containing fractions were combined and lyophilized. About 140 mg of protein were recovered.

Isolation of Connecting Peptide Fraction from Tryptic Digest by Cm-cellulose Chromatography-The desalted protein from the preceding tryptic digest was dissolved in 10 ml of 0.01 M sodium citrate buffer, 7 M urea, at pH 5.00 and applied to a column, 18 x 240 mm, of Cm-cellulose equilibrated with the same buffer at 2”. After application, the sample was rinsed into the column with several volumes of buffer. As soon as a small breakthrough peak emerged, a linear NaCl gradient was started (total volume = 300 ml) and 3.6-ml fractions were collected (Fig. 3). The fractions designated A, B and C were collected, desalted by gel filtration, and lyophilized.

Gradient El&ion Chromatography of Cm-cellulose Fraction A- The desalted protein of Cm-cellulose Fraction A (34 mg) was dissolved in 2 ml of buffer and chromatographed at room tem- perature on a column, 10 x 200 mm, of DEAE-cellulose in 0.01 M Tris-HCl at pH 8.0. After application of the sample

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of March 10, 1971 Xteiner, Cho, Oyer, Terris, Peterson, and Rubenstein 1367

0 IO 20 30 40 50 $0 70 80 90

FRACTION NUMBER

r

FIG. 3. Gradient elution chromatography on Cm-cellulose of the desalted tryptic digest of the bovine proinsulin intermediate fraction. Fractions designated A, R, and C were combined as shown by the heavy bars. (See “Materials and Methods” for procedural details.)

BOVINE INTERMEDIATE-TRYPTIC PEPTIDES

f ; .2 E

E . * lo w u 5

! m

. rjrod$/

_*-- _---

m .I . _---

8 _--- -_--

_----

: __-- -_---

t

,A-3, , > ) o(.:,7cj f-l A-f

16 18 20 22 24 26 28 30 32 34 36 38

FRACTION NUMBER

FIG. 4. Gradient elution chromatography on DEAE-cellulose of the desalted Fraction A from the preceding column chromatog- raphy (Fig. 3). Fractions designated A-f, Ad, and A-S were combined as shown by the heavy bars. (See “Materials and Meth- ods” for procedural details.)

a 0 to 0.2 M NaCl gradient was started and 1.9-ml fractions were collected. Three overlapping peaks eluted between 0.7 and 0.1 M NaCl (Fig. 4). Fractions designated A-l, A-2, and A-3 were combined, desalted on Sephadex G-15 and lyophilized. Recovery was about 80% of the starting material. Electro- phoretic analysis of the starting material and the three fractions obtained is shown in Fig. 5 and their amino acid compositions are compared in Table I. The purification procedure is sum- marized in Fig. 6.

Isolation of C-Pep&k from Bovine Pancreas-Frozen adult bovine pancreas (1.5 kg) was chopped into l-cm cubes and blended in batches with ice-cold acid-ethanol (4500 ml of 95% ethanol-90 ml of concentrated HCI) in a Waring Blendor for 30 sec. One-fifth volume of water was then added and the blend- ing continued for 10 to 20 sec. The extracts were combined and

PAPER ELECTROPHORESIS 30% FORMIC ACID 01

1 2 3

CM-23 FRACTION A 000 DE-23 A-l

0

;.“\ :.:

DE-23 A-2

DE-23 A-3

BOVINE INSULIN (Reference)

RIGI

1

N

4

FIG. 5. Bovine intermediate tryptic peptides. Diagrammatic representation of the results of paper electrophoresis of Cm-cellu- lose Fraction A (Fig. 3) and its DEAE-cellulose subfractions (Fig. 4). Approximately 1OOrg of each fraction dissolved in 30% formic acid were applied to a sheet (15 X 35 cm) of Whatman No. 3MM paper moistened with 30% formic acid. Electrophoresis was carried out at room temperature for 6 hours at 5 volts per cm. The dried electrophoretogram was stained with ninhydrin and the color was developed for 24 hours at 30”. The differences in mobil- ity between peptide spots 1, B, and S represent a difference of 1 basic-residue per molecule of peptide.

TABLE I Amino acid composition of connecting peptide fractions isolated

from tryptic digests of bovine proinsulin intermediate fraction

Glutamic acid Proline Glycine Alanine Valine Leucine

:sidues/moL

5.80 4.15 7.55 3.03 2.09 3.01

5.70 6.10 5.80 6 3.74 3.90 4.10 4 7.70 7.60 7.60 8 2.92 3.00 2.98 3 2.15 2.08 2.06 2 3.08 3.12 3.18 3

Lysine 0.42 Arginine 0.28

Proinsulin se- quence

1.13 1.0

PWP60

NH2 terminus Glu

--

re

-

CM-23, Fraction A

DE-23

A-l ) A-2 / A-3

0.56 0.26

PWP59 PWP58

Glu 4+ Arg 1+

Trace 0

PWP58

Glu

1 ‘heory”

-

-0.4 <0.4

(1 Based on known composition of bovine proinsulin connecting segment. The peptides having basic residues are assumed to arise almost entirely from Form II of the intermediate fraction (see Fig. 1). The cleavage of the carboxyl-terminal arginine to convert Peptide P33-PGo to P33-P59 depends on the length of tryptic di- gestion. The presence of arginine in Fraction A-2 is only partly due to cross-contamination with Fraction A-l, i.e. subtractive Edman degradation revealed that about two-thirds of the argi- nine was amino-terminal, indicating that Peptide Pz~-Ps~ com-

stirred in the cold room for several hours. After centrifugation prises about 15% of Fraction A-2

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1368 Bovine Proinsulin C-Peptide Vol. 246, No. 5

CRYSTALLINE BOVINE INSULIN BOVINE PANCREAS

I Gel Filtration

CRUDE PROINSULIN FRACTION

I CM-Cellulose Chromatography ,,,,,,E&i:T

I DEAE - Cellulose Chromatography

PURE INTERMEDIATE FRACTION (Fig. 1) +

I

SUPERNATANT PRECIPITATE

Trypsin Digestion

I

CM -Cellulose Chromatography 1 %%.Et;,.

(discard)

,

Tak C (Insulin)

SUPERNATANT PRECIPITATE

Peak A Peak B (discard) 1

Dissolve in IM Acetic Acid

I

DEAE Chromatography SEPHADEX G-50 (IM Acetic Acid)

I Insulin Fraction

I I BIOGEL P - 30 (3 M Acetic Acid)

Peak A, Peak As Peak As I Insulin Fraction

I Desalt

C - PEPTIDE

(Pss - %s)

Peak A Desalt

r-

CM -CELLULOSE COLUMN OOI M Citrate, pH-5.0

7 M Urea

ELECTROPHORESIS (30% Formic Acid)

1 Fraction a ELECTROPHORESIS (1.2 M Pyridine Acetate)

I

pH-6.5

Fraction a,

CELLULOSE COLUMN ( butanol- 1 -pyridine -acetic acid -HsO

I 60:40:12:48v/v )

C-PEPTIDE

FIG. 6. Summary of the extraction and purification procedures for the proinsulin C-peptide (left) and the pancreatic C-peptide (right). (See “Materials and Methods” for procedural details.)

BOVINE PANCREATIC EXTRACT

1 Kg Sephadex G - 50 IM Acetic acid

INSULIN PEAK

60 70 60 90 100 110 120 130 140 150 160 170

FRACTION NUMBER

FIG. 7. Elution profile of partially purified acid-ethanol extract of 1 kg of bovine pancreas from a column (8 X 100 cm) of Sephadex G-50 in 1 M acetic acid. The fractions included in the heavy bar contained both insulin and C-peptide as well as several other peptides. (See “Materials and Methods” for procedural details.)

at 600 x g for 20 min the supernatant fluid was adjusted to pH 8.0 with concentrated NHdOH and the resultant precipitate was discarded by centrifugation. The pH of the supernatant fluid was adjusted to pH 5.3 with 6 N HCl, using methyl red as an indicator, and 1 volume per 40 volumes of 2 M NHI- acetate (pH 5.3) was added. Insulin, proinsulin, C-peptide, and other proteins were precipitated overnight at 3” by addition of 2 volumes of absolute alcohol and 4 volumes of diethyl ether. The precipitate was collected by centrifugation at 600 X g for 20 min and dissolved in 250 ml of 1 M acetic acid.

Gel filtration was initially carried out with one-third (500 g), and subsequently with two-thirds (1 kg), of the total extract over a column (8 X 100 cm) of Sephadex G-50 (fine) eluted with 1 M acetic acid. The elution profile of the 1 kg eq of extract is shown in Fig. 7. Paper electrophoresis in 30% formic acid of selected fractions from the region of tubes 110 through 180 identified the ultraviolet absorbing component at Fraction 144 as mainly insulin, and this was confirmed by polyacrylamide electrophoresis at pH 8.4 (20) and by immunoassay (21). Electrophoresis showed a second ninhydrin-positive Pauly- negative component that had the same mobility as the proin- sulin C-peptide. It was distributed through the insulin frac- tions, being more abundant in the tubes immediately after the insulin peak, indicating a slightly greater Vi/V0 (22) than insulin. (There was no evidence that the peptide associated with insulin under the conditions used for gel chromatography, the elution positions of either component alone being the same as in mixture.) The tubes containing insulin and the region immediately following it (tubes 132 to 160) were then combined and lyophilized.

Further purification of this material was carried out by a second gel filtration on a column (5 X 105 cm) of Bio-Gel P-30 eluted with 3 M acetic acid. This column gave an improved separation of several higher molecular weight impurities as shown in Fig. 8. The insulin-containing peak (designated 4) was identified and collected by lyophilization. About 250 mg of dry protein were obtained. Gradient elution chromatography of this material was carried out on a column (18 X 250 cm) of Cm-cellulose at pH 5.0 as described above for the tryptic digest of the proinsulin intermediate fraction. As the 7 M urea buffer

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of March 10, 1971 Steiner, Cho, Oyer, Terris, Peterson, and Rubenstein 1369

1.2

i

BIOGEL P-30 INSULIN

-I 3M Acetic Acid

20 40 60 80 100 120 140

FRACTION NUMBER

FIG. 8. Repeat gel filtration of insulin-containing fractions from the Sephadex G-50 column on a column (5 X 100 cm) of Bio-Gel P-30 in 3 M acetic acid. The tubes containing Fractions 1 through 4 were combined as shown. Fraction 4 contained insulin and C-peptide.

precludes detection with ninhydrin, a few micrograms of ribo- flavin were added to the extract dissolved in the buffer (0.01 M

sodium citrate, pH 5.0-7 M urea) to enable identification of the breakthrough volume by fluorescence. Peaks A, B, and C were collected as shown in Fig. 9, desalted over Sephadex G-15 (Peak A) or G-25 (Peaks B and C) in 0.1 M acetic acid, and lyophilized. The peptides were detected after gel filtration by spotting small aliquots of each fraction on numbered paper grids which were dried and developed with ninhydrin aerosol (Ninspray, Sigma). Fraction A, containing C-peptide and other peptide contaminants, weighed 110 mg. Fraction C contained 90 mg of insulin.

Further pur%cation of Fraction A was carried out by paper electrophoresis as shown in Fig. 10. The initial electrophoresis in 30% formic acid separated a major component (CY) having the same mobility as tryptic Peptide A-3, from several com- ponents migrating more rapidly toward the cathode. After ascending elution from the paper with 50% acetic acid, Frac- tion A-cr was further resolved into four components by electro- phoresis in 1.2 M pyridine acetate at pH 6.5 (Fig. 10). The major ninhydrin-positive component, having the same mobility as tryptic Peptide A-3, and the other three spots were eluted from the paper with 50% acetic acid and submitted to amino acid analysis (Table II). The C-peptide (Fraction A-al) at this stage contained only small amounts (-1%) of contaminating amino acids, mainly aspartic acid, threonine, and serine. These were almost completely eliminated by partition chromatography on a column (0.9 x 55 cm) of formylated cellulose eluted with l-butanol-pyriclme-acetic acid-water (60 :40: 12 :48, v/v). Frac- tions of 1.07 ml were collected. Small aliquots from each fraction were spotted on numbered paper grids and developed with the ninhydrin spray reagent. The single peak was arbi- trarily divided into four regions, the fractions were combined, and aliquots from each were submitted to amino acid analysis. The three early fractions from the peak contained almost no aspartic acid, serine, or threonine (Table II), whereas the last fraction to be eluted was heavily contaminated with peptides containing these amino acids. The fractions having minimal contamination ( <O.l %) were combined and utilized for further characterization and sequence analysis. The molar yields of insulin and C-peptide were found to be nearly identical, being about 10 pmoles per kg of fresh pancreas for each. The isola- tion procedure is summarized in Fig. 6.

I :

1.6-

E

1.4. C-PEPTIDE

?A 1.2- 8 $ IO-

, /’

.2- /’

/’ A /’ B C

04 0 ,o 20 30 40 50 60 70 80 90

; <

10 6 I

3 s I I

.05 1

0

FRACTION NUMBER

FIG. 9. Gradient elution chromatography on Cm-cellulose of the insulin peak from gel filtration of bovine pancreas extract (Fig. 8). Fractions designated A, B, and C were combined as

shown by the heavy bars. Absorbance of Peak A (compare with Fig. 3) was due to added riboflavin and to impurities present in the pancreatic preparation. Peak B contained several com- ponents but no deoctapeptide insulin.% (See “Materials and Methods” for procedural details.)

30% FORMIC ACID

Y Pa PANCREAS FRACTION

A :::::(:j:::(:;:;

0

TRYPTIC PEPTIDE A-3 $

I I I +I

TRYPTIC PEPTIDE A-3

+

FICA 10. Paper electrophoresis of pancreatic C-peptide frac- tions. Diagrammatic representation of paper electrophoretic purification of pancreatic C-peptide preparation (Fraction A of Fig. 9) in two systems. Upper panel, 30% formic acid; lower panel, 1.2 M pyridine acetat.e, pH 6.5. The solid black spot repre- sents the pancreatic C-peptide spot while the hatched spot repre- sents the proinsulin C-peptide spot. (See “Materials and Meth- ods” for procedural details.)

Chywwtryptic Digestion and Peptide Mapping-Approximately 1.0 mg (0.4 pmole) of the pancreatic (a& and proinsulii (A-3) C-peptides were dissolved in 0.2 ml of 0.02 M Tris buffer and adjusted to pH 8.4 with 1 N NaOH. Sufficient stock solution

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1370 Bovine Proinsulin C-Peptide

TABLE II

Vol. 246, No. 5

Acidic and neutral a.mino acid composition of pancreatic C-peptide and contaminants separated by pyridine acetate electrophoresis

Aspartic acid. .............. .............. Threonine ................................... Serine ...................................... Glutamic acid ............................... Proline .................................... Glycine ..................................... Alanine ..................................... Valine ...................................... Isoleucine .................................. Leucine ..................................... Tyrosine ................................... Phenylalanine. .............................. Amino sugar. ............................... Lysine ...................................... Arginine ...................................

0.03 0.47 0 0.14 0.03 0.45

23.9 (6.2)b 22.2 (5.8)b 15.02 (3.9) 14.4 (3.8) 29.7 (7.7) 29.6 (7.7) 10.8 (2.9) 12.2 (3.2)

8.0 (2.1) 8.3 (2.2) 0 0

10.9 (2.9) 12.2 (3.2) 0 0 0 0 0 0 0 0 0 0

1.7 24.8 0.6 3.3 0.9 4.4

23.1 5.5 14.1 7.7 28.2 7.1 10.7 3.4

7.4 1.2 0 1.3

11.6 3.4 Trace Trace Trace Trace

0.6 37.8

Percentage of total starting material. ....... 78 13 4.5 -

C-Peptide

-

20.8 5.2

11.6 2.0 5.1 3.2 2.3 0.9 0.2 0.7 0.1

Trace 48.0

4.5

a Fraction cyi after partition chromatography on formylated cellulose column (see “Materials and Methods” for procedural details) b Numbers in parentheses represent residues per mole of C-peptide. See Table I for composition of bovine proinsulin C-peptide.

TABLE III

Chymotryptic peptides

Peptides Amino acid composition

A. Proinsulin C-peptide (A-3) a. 270 ehymotrypsin digest

P-l, Residues 13-26. P-2, Residues 11-26. . P-3, Residues l-10. P-4, Residues 1-12. . .

B. Pancreatic C-peptide (CQ) a. 2% chymotrypsin digest

P-l, Residues 13-26. . P-2, Residues 11-26. P-3, Residues l-10.. P-4, Residues 1-12. .

b. 5% chymotrypsin digest F2T1, Residues 22-26 F2T2, Residues l-6. F2T3, Residues 13-21. F4, Residues 11-12.

Glu 2.1(2), Pro 2.9(3), Gly 5.7(6), Ala 1.9(2), Leu 1.0(l) Glu 3.1(3), Pro 3.1(3), Gly 6.0(6), Ala 1.8(2), Leu 2.0(2) Glu 3.2(3), Pro 1.0(l), Gly 2.1(2), Ala 0.9(l), Val 2.0(2), Leu 0.95(l) Glu 4.0(4), Pro 1.1(l), Gly 2.0(2), Ala 1.0(l), Val 1.9(2), Leu 1.9(2)

Glu 2.0(2), Pro 2.9(3), Gly 5.9(6), Ala 1.9(2), Leu 1.0(l) Glu 3.0(3), Pro 3.2(3), Gly 6.0(6), Ala 1.9(2), Leu 2.0(2) Glu 3.0(3), Pro 1.0(l), Gly 2.1(2), Ala 1.0(l), Val 1.9(2), Leu 1.0(l) Glu 4.0(4), Pro 1.0(l), Gly 2.0(2), Ala 1.0(l), Val 2.0(2), Leu 2.1(2)

Glu 2.0(2), Pro 1.9(2), Gly 1.0(l) Glu 2.8(3), Pro 1.0(l), Gly 1.1(l), Val 0.8(l) Glu 0.2(O), Pro 1.0(l), Gly 5.0(5), Ala 2.0(2), Val 0.1(O), Leu 1.05(l) Glu 1.0(l), Gly 0.15(O), Ala 0.1(O), Leu 1.1(l)

of cr-chymotrypsin dissolved in 0.001 N HCl (2.0 mg per ml)

was added to give a final enzyme to substrate ratio of 1:50. The sealed tubes were incubated 20 hours at 37” in a water bath. The reaction was stopped by addition of glacial acetic acid (1: 10, v/v, reaction mixture) and the samples were lyophi- lized. Mapping of peptides was performed on thin layer cellu- lose plates (20 X 20 cm) (Eastman Catalogue Number 6064). Approximately 50 pg of digest, dissolved in 30% formic acid, were applied to a thin layer plate moistened with 30% formic acid. Electrophoresis was carried out for 5 hours in a field of 6 volts per cm. After drying the plates, ascending chromatog- raphy was carried out in I-butanol-pyridine-acetic acid-water (60:40: 12:48, v/v). The dried plates were stained with nin-

hydrin spray reagent and were photographed after optimal color development at room temperature (48 to 72 hours).

For preparative purposes the peptides were resolved into four major components by electrophoresis on Whatman No. 3MM paper in 1.2 M pyridine acetate buffer, pH 6.5. After elution from the paper with 50% acetic acid, these were designated P-l, P-2, P-3, and P-4 in order of their increasing anodal mobili- ties. Further purification, where necessary, was carried out by paper electrophoresis in 30% formic acid or by thin layer chroma- tography on cellulose sheets in l-butanol-pyridine-acetic acid- water (60:40: 12:48, v/v). The compositions of the four major chymotryptic peptides obtained are given in Table III.

Further cleavage of the pancreatic C-peptide was achieved

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of March 10, 1971 Steiner, Cho, Oyer, Terris, Peterson, and Rubenstein 1371

by incubating it for 20 hours at a concentration of 5 mg per ml with 5% (w/w) chymotrypsin in 0.05 M Tris-HCl buffer, pH 9.0, containing 3 mu CaC12. Paper electrophoresis of this di- gest was carried out for 10 hours at 5 volts per cm in 30% formic acid. Four ninhydrin-positive fractions migrating to- ward the cathode were designated Fl to F4 in order of increasing electrophoretic mobility. Thin layer chromatography of the eluted fractions on cellulose sheets in I-butanol-pyridine-acetic acid-water (60:40:12:48, v/v) revealed three major com- ponents in both Fractions Fl and F2, and only one major component in Fractions F3 and F4. Each major component was separated by thin layer chromatography and eluted from the cellulose with 50% acetic acid. The amino acid compositions of these peptides are given in Table III.

Digestion with Carboxypeptidase A-Carboxypeptidase A crystals were washed with water and dissolved as described by Ambler (23). C-Peptide (300 pg) was dissolved in 100 ~1 of 0.05 M Tris-buffer containing 0.5% NaCl and the pH adjusted to 8.5 with 0.1 N NaOH. Twenty microliters of carboxypepti- dase stock solution (5 mg per ml) were then added to give a final molar ratio of enzyme to substrate of 1:30. A small aliquot was removed and quenched with 1 drop of glacial acetic acid before adding the enzyme. Incubation was carried out at 37”, and aliquots were removed for ammo acid analyses at various times up to 11 hours. Enzyme containing blanks were similarly incubated.

Other Procedures-Stepwise degradation of peptides was car- ried out by a semimicro adaptation of the direct Edman pro- cedure (25) using trifluoroacetic acid at the cleavage step (8, 24, 25). About 0.2 pmole of peptide was usually used. Phenyl- thiohydantoin derivatives were identified by thin layer chroma- tography on silica gel thin layer plates as described by Jeppsson and Sjijquist (26). In most instances NH&erminal residues were also detected at each step by dansylation of a small aliquot of peptide (0.005 pmole). Dansyl derivatives were identified by thin layer chromatography on polyamide plates according to the procedure of Woods and Wang (27). Amino acid analyses were carried out on samples of 0.01 to 0.1 pmole of peptide after hydrolysis in degased 6 N HCl in small glass tubes sealed in a vacuum and incubated at 110 f 1” for 20 to 30 hours. Some analyses (Table I) were performed on a single column Technicon AutoAnalyzer by the 14-hour procedure essentially as described by Piez and Morris (28). Later analyses (Tables II and III) were performed on a Beckman-Spinco model 120 C using the standard two column system (29) with recorder scale expansion and electronic integration.

Immunoassays were carried out using the double antibody procedure of Morgan and Lazarow (21) as modified by Ruben- stein et al. (19).

RESULTS AND DISCUSSION

Isolation of C-Peptide from Typsin Digests of Bovine Proinsulin Intermediate Fraction-Earlier experiments had indicated that the intermediate fraction, like proinsulin, could be converted by trypsin to an insulin-like component that was identified as dealanine insulin (6, 8, 11, 30). In preliminary experiments it was possible to resolve the major tryptic transformation products in one dimension by paper electrophoresis in 30% formic acid and thus to observe the relative rates of cleavage of various bonds in proinsulin. Incubation of intact proinsulin (10 mg per ml) with 1% trypsin (w/w) at pH 8.2 (0.1 M Tris-

HCl) resulted in rapid liberation of the polypeptide P33-PG0 by cleavage at positions PZ2-PZ3 and P60-P~l within 2 to 3 min, leaving insulin bearing 2 arginine residues at the COOH terminus of the B chain. More prolonged incubation led to cleavage at Bzs-Bso (Lys-Ala) to yield dealanine insulin, and slower cleavage between P31-P32 and P59-Ps0 to liberate free arginine, Ala-Arg, and Peptides P33-Pso or P33-P59 having NHz-terminal glutamic acid and COOH-terminal lysine or Lys-Arg depending on the length of the tryptic digestion. Due to the specificity of tryp- sin, cleavage of the Gln-Lys sequence (Ps-Pb9) of intact pro- insulin was not observed, and thus the C-peptide was not pro- duced.

Although the same bonds are cleaved in the tryptic digestion of the proinsulin intermediate fraction as in intact proinsulin, this fraction also gives rise to a connecting peptide fragment which lacks all 4 of the terminal basic residues, i.e. the C-peptide. It is clear from timed studies of the tryptic digestion that the most rapid cleavage occurs at the Arg-Gly (Pso-PGr) and Arg-Glu (P32-P33) sequences. Thus, incubation of the intermediate fraction with 1 to 2% trypsin by weight liberates mainly two peptides, P22-Pss and Pzr-Pso, almost quantitatively within the first few minutes. (In Form I, cleavage at Arg-Arg (P,i-P,,) occurs only in about 5% of the material to yield Peptide Pas-P58 (Table I).) The diarginyl insulin remaining from Form I of the intermediate fraction (see Fig. 1) aggregates to form a turbid white suspension due to its much higher isoelectric pH compared to that of insulin. As further tryptic cleavages remove these arginine residues from the insulin during the ensuing 15 to 20 min the products are more soluble and the turbidity clears. During this interval, cleavage also occurs between Lys-Arg (Pss-PsO) of Peptide Pla-Pso to yield Peptide Ps3-Ps9.

Thus, three peptide fractions, A-l, A-2, and A-3 (Table I), can be isolated from the tryptie digests of the intermediate forms, whereas with intact proinsulin tryptic cleavage releases only Fractions A-l and A-2. It therefore can be concluded that Fraction A-3, the C-peptide, arises from cleavage of Form I of the two intermediates, i.e. the form in which both COOH- terminal lysine and arginine (P-59 and P-60) are already absent. Since the rate of cleavage of the Lys-Ala sequence of the B chain (BZ9-Ba0) is evidently increased in proinsulin by the presence of the Arg-Arg sequence at P31-P32 (31), it is likely that the over-all rate of cleavage at this position in the intermediate fraction, 40% of which lacks the Arg-Arg sequence, will differ from that in intact proinsulin. The kinetic aspects of the trypsin digestion of these various molecular species will be examined in greater detail in subsequent reports.

In previous studies it was noted that the bovine proinsulin intermediate fraction is not bound to Cm-cellulose columns at pH 5.5 in 0.01 M sodium citrate-7 M urea (6). Since the various connecting peptides released by trypsin all have a strong net anionic charge above pH 4.3 due to the presence of the four glutamic acid side chains, it was predicted that these likewise would not bind to such a column. However, to ensure the binding of any residual intermediate fraction and of the insulin, dealanine insulin, and deoctapeptide insulin produced by tryp- sin, the pH of this chromatographic system was lowered to 5.0. Under these conditions the peptides were not bound and the various insulin derivatives could be efficiently separated by means of a salt gradient (Fig. 3).

The three tryptic peptide fractions were resolved according to the number of basic residues either by chromatography on

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Bovine Proinsulin C-Peptic% Vol. 246, No. 5

DEAE-cellulose at pH 8.0 as shown in Fig. 4, or by paper eleotrophoresis in 30% formic acid as shown in Fig. 5. Although the preparative DEAE-cellulose column chromatography did not completely separate the three fractions, the results of oom- positional studies shown in Table I confirm that the three peptides are identical with respect to their content of acidic and neutral amino acids, and the amounts of these correspond with the composition of the connecting segment of bovine proinsulin (11, 12). Aside from Fraction A-2 which contained 8. small proportion of material having NH*-terminal arginine, dansylation studies revealed only glutamic acid in all three fractions. Thus it is clear that the basic residues are indeed carboxyl-terminal in location, as with all tryptic peptides arising fmm internal sequences in proteins.

lsolaltin of Pancreatic C-PeplideBiosynthetie studies utiliz- ing isolated rat islets of Langwham indicated that the conneot- ing peptides liberated in uiuo were extracted quantitatively with insulin through the stage of isoelectric precipitation at pH 5.3 with organic solvents, but were not salted out with insulin by aqueous 15% N&l nor retained with insulin during its crystalliiation (32). Accordingly, the acid-ethanol extracts of bovine pancreas were purified only through the isoelectric precipitation step (33). Considerable further purification w&s then achieved by gel filtration on a large column of Sephadex G-50 equilibrated with 1 .O M acetic acid (Fig. 7) followed by rechromatography on a smaller column of Bio-Gel P-30 (Fig. 8). From earlier biosynthetic experiments and studies of the tryptio products of leucine-labeled rat proinsulin (12, 32), it was ex- pected that the connecting peptides would elute almost coinei- dently with the insulin peak. Since the insulin peak can usually be located by optical density measurements at 275 mp, this procedure w&s adopted for lo&ion of both insulin and the connecting peptides. Except for relatively small amounts of contaminating peptides the insulin fraction w&s comparable in composition to the tryptic digests of intact or intermediate proinsulins and therefore could also be purified by Cm-cellulose chromatography. As shown in Fig. 9 essentially similar results were obtained, the more acidic connecting peptide appearing with the breakthrough volume.

MOLARITY

Fro. 11. Displacement of ‘a’I.bovine proinsulin from & guinea pig bovine proinsulin antiserum (1:250,ooO dilution) by bovine proinsulin snd by the pancreatic C-peptide (0) or the proinsulin C-peptide (0). No dierence in immunological behavior of the two peptides w&4 detected.

Further purification of the peptide was most conveniently carried out by paper eleetrophoresis (Fig. 10). The advantage of this method is that it allows comparisons with known peptide standards. It can be seen from the upper panel of Fig, 9 that the Cm-Fraction A consisted mainly of peptide corresponding in mobility to tryptic Peptide A-3, the C-peptide. However, 8s noted previously, this material contained appreciable amounts of amino acids not present in the C-peptide, 8s well as a con- siderable amount of an amino sugar that w&s tentatively identi- fied as glucosanine (17). Electrophoresis in 1.2 M pyridine acetate, pII 6.5, separated three slowly migrating components which comprised about 20% of the total peptide fraction. The spot A-a (Fig. 10) which corresponded in mobility with the tcyptic C-peptide (A-3) had the expected amino acid composition except for about 1 to 2% contamination with peptides con- taining aspartic acid, serine, and threonine (Table II). Further removal of these impurities to below 0.1% was achieved either by thii layer chromatography on cellulose sheets, or in the same solvent system over a partition column of formylated cellulose powder. In the latter system the aspartie acid-, threonine-, and swine-containing contaminants eluti after the C-peptide, whereas on thin layer cellulose plates they migrated slightly more rapidly than the C-peptide. Both the pancreatic and tryptic C-peptides had the same RP value in the thin layer S@,SI&

The results of the studies presented thus far indicated that pancreatic Peptide A-w is identical with the proinsulii C-pep- tide with respect to its exclusion volume on gel filtration, its mobility in two electrophoretic systems, its R, on thii layer partition chromatography, and its identical amino acid oomposi- tion and amino-terminal residue. Further indications of its identity were apparent in its immunological crossreactivity with bovine proinsulin antisera. As shown in Fig. 11, both the tryp- tic and pancreatic C-peptides were equally effective in displacing ‘8’I-labeled bovine proinsulii from anti-proinsulin antisera. This reaction is hiihly specific for the bovine C-peptide, as neither porcine nor human C-peptide react in this system, even at 1000.fold higher concentration.

Proof of Identity of Pan&tic and Tryptie C-Pep&k-In order to establish the identity of the primary structure of the

FIG. 12. Comparison of peptide maps of the bovine proinsulin C-peptide and the pancreatic C-peptide. The few heavy spots arranged more or less vertically in each photograph correspond to chymotryptic Peptides P-l, P-2, P-3, and P-4 (frmn below upward). The more rapidly cathodally migrating component is the dipeptide Glu-Leu (Residues 11 and 12).

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of March 10, 1971 Steiner, Cho, Oyer, Terris, Peterson, and Rubenstein 1373

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

GLU-VAL-GLU-GLy-PRO-GLN-VAL-GLY-ALA-LEU-GLU-LEU-ALA-GLY-GLY-PRO-GLY-ALA-GLY-GLY-LEU-GLU-GLY-PRO-PRO-GLN

,“,ac, _y --A --A --A --A --L - 4

t- 1F2T2 p,---L--------- 4

p21- --& - - - I

a s - --L F2Tlr- - -

-

p3 - --1 s - --A a -1 , p4 A --A A A --> - - - - --a - 2

F4. F2T31-

L-- L-- L--- -

KEY: - BOTH DERIVATIVES SEEN --2. PTH ONLY SEEN

- DANSYL ONLY SEEN

- CPASE A

FIG. 13. Schematic summary of the amino acid sequence determination of the bovine pancreatic C-peptide and chymotryptic pep- tides. Identical results were obtained with the proinsulin C-peptide which also was carried through these procedures as a reference. (See “Materials and Methods” for procedural details.) CPASE A, carboxypeptidase A; PTH, phenylthiohydantoin. Each arrow in- dicates one step of Edman degradation.

pancreatic C-peptide with the known structure of the proinsulin C-peptide both purified peptides were subjected to amino acid sequence analysis by direct Edman degradation of the intact peptides and their chymotryptic fragments. Prolonged diges- tion of the peptides with 2% chymotrypsin (w/w) resulted in cleavage at the first 2 leucine residues (positions 10 and 12) but not at the 3rd leucine (position 21) to give rise to the four major peptides shown in the peptide maps in Fig. 12. The amino acid compositions of the four chymotryptic peptides derived from the pancreatic peptide were identical with those derived from the corresponding proinsulin peptide as shown in Table III. Since Peptides P-3 and P-4 comprised the amino-terminal 10 and 12 residues of the C-peptide, respectively, these peptides were combined for Edman degradation.

In all cases the pancreatic and proinsulin peptide fragments were degraded in parallel and the results compared. Peptide P-l was degraded through 12 steps, while Peptide P-2 was de- graded only 5 steps to establish the overlapping Glu-Leu sequence at positions 11 and 12 of the intact peptide. The intact peptide was also degraded through 7 steps. The results are summarized schematically in Fig. 13. With two exceptions the phenylthiohydantoin or dansyl derivative, or both, present alone or in largest amount was identical with the residue at the corresponding region of the bovine proinsulin sequence reported earlier (12, 34). The exceptions were in Peptide P-l at position 6 where alanine was more abundant than glycine, and at position 8 where glycine rather than alanine was more abundant. The presence of alanine rather than glycine at position 6 was con-

firmed by repeat degradation of these peptides utilizing the subtractive method as well as direct phenylthiohydantoin and dansyl identification at this position.

To further substantiate this difference in the sequence, a new chymotryptic digest of the peptide was prepared at a slightly higher pH with a higher ratio of chymotrypsin to substrate (5%, w/w) and with added calcium ions. Under these conditions cleavage occurred at leucine (Residue 21) and also at glutamine (Residue 6) as well as at the other leucines (Residues 10 and 12) to liberate Peptides F2T1, F2T2, and F2T3 (Fig. 13 and Table III). Peptide F2T3, Residues 13 to 21 of the C-peptide, was

TABLE IV

Digestion of Peptide F2Td with carboxypeptidase A

Enzyme blank

Peptide

-

_- min

120

660

0 0

mJ4moles

0 0

0 0.08

0 0 0 0 20 Trace Trace 5.34 60 0.14 0.07 5.25

120 0.30 0.14 5.77 660 1.69 0.47 5.930

Amino acids released

Glycine AlaCIle

a Total peptide per aliquot analyzed is approximately 6 rnp- moles.

submitted to carboxypeptidase digestion. The results (Table IV) clearly demonstrate that only leucine was released rapidly and quantitatively. At all intervals the release of alanine was very slow and it was always accompanied by at least 2 eq of glycine, in keeping with the proposed sequence (Fig. 13). The analogous peptide from the proinsulin C-peptide gave essentially identical results with carboxypeptidase.

Peptide F2Tl provided the carboxy-terminal pentapeptide and was degraded by the Edman procedure to confirm this portion of the sequence including the amide of glutamic acid at position 26, as shown in Fig. 13. Carboxypeptidase digestion of the two intact C-peptides failed to liberate free amino acids from either, in keeping with the COOH-terminal Pro-Pro-Gln sequence (23).

It is clear from these results that both peptides have identical ammo acid sequences, as equivalent results were obtained throughout these sequence studies. Although it is possible that the differences between these and the earlier sequence results were due to microheterogeneity in the bovine C-peptide, there is no indication of this from the carboxypeptidase data presented here. It seems more likely that the unusual abundance of gly-

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

1374 Bovine Proinsulin C-Peptide Vol. 246, No. 5

tine in this region of the sequence caused difficulties in the in- terpretation of some of the results obtained in the earlier se- quence studies (12, 34).

The conclusion that the pancreatic C-peptide is identical with the C-peptide of proinsulin is important, not only from the standpoint of the insight it provides into the normal biochemical converting mechanism in the pancreas, but also because it pro- vides a new and more abundant source of material for structural studies of proinsulins from a wide variety of species. Thus, in preliminary studies we have isolated an analogous peptide fraction from porcine pancreas that has the same composition and properties similar to the C-peptide portion of porcine pro- insulin2 Furthermore our preliminary results with bovine pancreas extracts (17) have been confirmed and extended to porcine extracts by Sundby and Markussen (35) who have uti- lized side fractions from the commercial production of bovine and porcine insulin for preparation of highly purified C-peptide.

Significant amounts of peptides corresponding to tryptic peptides A-l and A-2 (Table I) and having additional COOH- terminal lysine and arginine residues were not found in the bovine pancreatic extracts, although levels of a few per cent would not necessarily have been detected. Thus, the major form of free connecting peptide found in the pancreas is the peptide lacking all 4 terminal basic residues. It was for this reason that the designation C-peptide was assigned to this peptide rather than to either of the tryptic peptides or to the entire proinsulin con- necting polypeptide region (17).

Certain properties of the cleavage mechanism in viva may be deduced from these findings. Thus a trypsin-like endopeptidase could serve to open the polypeptide chain of proinsulin at the Arg-Gly sequence at the beginning of the A chain and at the Arg-Glu sequence just beyond the COOH terminus of the B chain. These cleavages occur readily in the presence of pan- creatic trypsin, to which these sequences are highly sensitive. However, trypsin will not cleave COOH-terminal to alanine, the normal carboxyl-terminal residue of the B chain of bovine and porcine insulin. Cleavage carboxyl-terminal to this residue could be accomplished by an exopeptidase similar to pancreatic carboxypeptidase B, which preferentially removes carboxyl- terminal basic residues from proteins or tryptic peptides. Thus the concerted action of enzymes similar to these two known pro- teases could produce the cleavages found in the intermediate components and lead to the liberation of insulin and of C-pep- tide. Novel peptidases having unique specificities adapted to this proteolytic function may exist, but this possibility seems less likely.

It is particularly noteworthy that equimolar amounts of C- peptide and insulin (10 pmoles per kg of fresh pancreas) were recovered. These findings are in accord with the proposed cleavage mechanism and lend support to the view that proinsulin is converted to insulin in the secretion granules where the de- tached C-peptide and insulin are then stored together (15, 16).

Aclcnowledgments-We are grateful to Drs. J. Schlichtkrull, F. Sundby, and J. Markussen of The Novo Company (Copen- hagen) for providing additional supplies of pancreatic C-peptide for completion of the amino acid sequence work. We wish also to thank W. P. Welbourne and W. S. Rudd for their assist-

2 W. S. Rudd and D. F. Steiner, unpublished data.

1.

2.

3. 4. 5.

6.

7.

8.

9.

STEINER, D. F., AND OYER, P. E., Proc. Nut. Acad. Xci. U. S. A., 67, 473 (1967).

STEINER, D. F., CUNNINGHAM, D. D., SPICELMAN, L., AND ATEN. B.. Science. 167, 697 (1967).

GRANT,‘~. +., AND REID,~K. B:M.,‘Biochem. J., 110.289 (1968). TUNG, A. K.,. AND YIP, C. C., Diabetologia, 4, 68 (1968) .- TRAKATELLIS. A. C., AND SCHWARTZ. G. P.. Nature. 226. 548

(1970) STEINER, D. F., HALLUND, O., RUBENSTEIN, A., CHO, S., AND

BAYLISS. C.. Diabetes. 17. 725 (1968). SCHMIDT, b. fi., AND ARE&, A:, Hippe-Seyler’s 2. Physiol.

Chem., 349, 1157 (1968). CHANCE, R. E., ELLIS, R. M., AND BROMER, W. W., Science,

161, 165 (1968).

10.

11.

RUBENSTEIN, A. H., AND STEINER, D. F., in R. CAMERINI- DAVALOS AND H. S. COLE (Editors), Proceedings of the Inter- national Symposium on Early Diabetes, Marbella, Spain, Academic Press, New York, 1970, p. 159.

CLARK, J. L., AND STEINER, D. F., Proc. Nat. Acad. Sci. U. S. A., 62, 278 (1969).

12.

13.

14.

15.

16.

NOLAN, C., MARGOLIASH, E., AND STEINER, D. F., Fed. Proc., 28, 343 (1969); NOLAN, C., MARGOLIASH, E., PETERSON, J. D.; AND STEINER, D. F., J. Biol. Chem.. in mess.

STEINER, D. F:, CLARK, J. L., NOLAN,-C., RUBENSTEIN, A. H., MARGOLIASH, E., ATEN, B., AND OYER, P. E., Recent Proar. Hormone Res.. 26, 207 (1969).

YIP, 6. C., AND LOGO&IE~OPO~LOS,‘J., PTOC. Nat. Acad. Sci. U. S. A., 62, 415 (1969).

SORENSON, R. L., STEFFES, M. W., AND LINDALL, A. W., En- docrinology, 86, 88 (1970).

RUBENSTEIN, A. H., CLARK, J. L., MELANI, F., AND STEINER, D. F.. Nature. 224. 697 (1969).

STEIN& D. F.,‘CL~RK, J: L., NOLAN, C., RUBENSTEIN, A. H., MARG~LIASH; E., ME~ANI, F., AND DYE& P. E., in E. CERAS; AND R. LUFT (Editors). Proceedinas of the 13th Nobel Sum- posium, Stockholm, John Wiley anYd Shns, Inc., New Y&k, 1969, p. 57.

17.

18.

CLARK, J. L., CHO, S., RUBENSTEIN, A. H., AND STEINER, D. F., Biochem. Biophys. Res. Commun., 36, 456 (1969).

WANG, S.-S., AND CARPENTER, F. H., J. Biol. Chem., 240, 1619 (1965).

19.

20.

21. 22 23.

RUBENSTEIN, A. H., WELBOURNE, W. P., MAHO, M., MELANI, F., AND STEINER, D. F., Diabetes, 19, 546 (1970).

ORNSTEIN, L., AND DAVIES, J. B., Disc electrophoresis, Distri- bution Products Industries. Rochester. New York. 1962.

MORGAN, C. R., AND LAZARO~, A., Diabetes, 12, 115 (1963). WHITAKER, J. R., Anal. Biochek., $6, 1950 (1963). AMBLER. R. P.. in C. H. W. HIRS (Editor). Methods in enzumol-

24.

25.

26.

27.

28. 29.

30.

31.

32. 33. 34. 35.

ante in various aspects of this work and Drs. C. Nolan and R. E. Chance for useful discussions.

REFERENCES

ogy, T&Z. XI; Academic Press, New York, 1967, p. 155. ” ERIKSSON, S., AND SJ~QUIST, J., Biochim. Biophys. Acta, 46,

290 (1960). BLOMBXCK, B., BLOMR~CK, M., EDMAN, P., AND HESSEL, B.,

Biochim. Biophys. Acta, 116, 371 (1966). JEPPSSON, J.-O., AND SJBQUIST, J., Anal. Biochem., 18, 264

(1967). WOODS, K. R., AND WANG, K.-T., Biochim. Biophys. Acta, 133,

369 (1967). PIEZ, L. A., AND MORRIS, L., Anal. Biochem., 1, 187 (1960). SPACKMAN, D. H., in C. H. W. HIRS (Editor), Methods in

enzymotogy, Vol. XI, Academic Press, New York, 1967, p. 3. STEINER, D. F., AND CLARK, J., Proc. Nat. Acad. Sci. U. S. A.,

60, 622 (1968). WANG, S.-S., AND CARPENTER, F. H., J. Biol. Chem., 244, 5537

(1969). CLARK, J. L., Ph.D. thesis, The University of Chicago, 1969. DAVO~EN, P. R., Biochim. Biophys. Acta, 63, 150 (1962). STEINER. D. F.. Neu Enal. J. Med.. 280. 1106 (1969). STJNDBY,‘F., AND MAR&JSSEN J., Hormone tietab: Res., 2, 17

(1970).

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Arthur H. RubensteinDonald F. Steiner, Sooja Cho, Philip E. Oyer, Susan Terris, James D. Peterson andIsolation and Characterization of Proinsulin C-Peptide from Bovine Pancreas

1971, 246:1365-1374.J. Biol. Chem. 

  http://www.jbc.org/content/246/5/1365Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/246/5/1365.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on May 20, 2018

http://ww

w.jbc.org/

Dow

nloaded from