Mode of activation and N-terminal sequence of subunit II in bovine procarboxypeptidase A and of porcine chymotrypsinogen C

82

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35346 MODE OF ACTIVATION AND N-TERMINAL SEQUENCE OF SUBUNIT II IN BOVINE P R O C A R B O X Y P E P T I D A S E A AND OF PORCINE CHYMOTRYPSINOGEN C

R. J. P E A N A S K Y * , D. G R A T E C O S , J. B A R A T T I AND M. R O V E R Y

Centre de Biochimie et de Biologic Moldculaire ~", C.N.R.S., Marseille (France) (Received N o v e m b e r 8th, 1968)

SUMMARY

In accordance with what is observed for bovine or porcine chymotrypsinogens A and B, the activation of porcine chymotrypsinogen C and of Subunit II in bovine procarboxypeptidase A involves the specific cleavage by trypsin of the first basic bond in the N-terminal region. The short chain thus formed contains 13 residues instead of 15. The two missing residues correspond to an apparently characteristic deletion at positions 12 and 13 . A comparison of the chains (N-terminal sequences) in six zymogens supports the view that bovine Subunit II is a chymotrypsinogen resembling more chymotrypsinogen C than chymotrypsinogens A and B. This suggests the existence in bovine and porcine pancreas of at least two related but distinct chymotrypsinogen lines,

INTRODUCTION

Subunit II of bovine proearboxypeptidase A-S6 and A-S5 (refs. I and 2) resembles in many respects the chymotrypsinogens A and B from bovine and porcine pancreas. It contains a half-cystine residue at the N-terminal position. Its activation requires low amounts of trypsin at o ° and appears to involve the cleavage of a single peptide bond 1. The resulting endopeptidase splits the synthetic ester substrates commonly used for chymotrypsin. However, these properties are also characteristic of porcine chymotrypsinogen C (ref. 3). In addition, the amino acid composition of chymotrypsinogen C and Subunit n are nearer each other than they are to the composition of chymotrypsinogens A and B (refs. 3 and 4). Their molecular weights appear to be somewhat larger 4 and the corresponding enzymes have a higher affinity for leucyl bonds 3. It has therefore been postulated3, 4 that chymotrypsinogen C and Subunit II represented in A b b r e v i a t i o n s u s e d : Z - G l y - P h e , c a r b o b e n z o x y g l y c y l - L - p h e n y l a l a n i n e ; A T E E , acetyl-Lt y r o s i n e e t h y l ester; D F P , diisopropylfluorophosphate. * P r e s e n t address: U n i v e r s i t y of S o u t h D a k o t a School of Medicine, Vermillion, S. D. 57069, U.S.A. ** P o s t a l a d d r e s s : C.N.R.S., C.B.M., 31 C h e m i n J o s e p h Aiguier 13, Marseille 9 °, France.

Biochim. Biophys. Acta, i 8 i (1969) 82-92

PROCARBOXYPEPTIDASE

A AND CHYMOTRYPSINOGEN C

83

porcine and bovine pancreas a line related to, but distinct from, the line leading to chymotrypsinogens A and B. This hypothesis has been confirmed by the study, described below, of the short chain (chain A) formed during the activation of the zymogens. MATERIALS AND METHODS

Preparation of columns Sephadex G-Ioo or G-5o (medium) was allowed to swell for 2 days in I M NaC] or 5 mM HC1, respectively. Equilibration of the packed columns was attained by passage of I 1 of the respective solutions through the colunms. DEAE-cellulose (Schleicher and Schuell, type 4 o, capacity 0. 9 mequiv/g) was stirred with a mixture of 0.5 M NaC1 and 0. 5 M NaOH, washed with water and then equilibrated in phosphate buffer (90 raM, p H 7.0). After pouring, the columns were washed with I 1 of the same buffer.

Enzyme assays Activation of Subunit I of bovine procarboxypeptidase A (leading to carboxypeptidase A) was carried out at 37 ° in phosphate buffer (5° raM, p H 7.o) with a i : 2 or 1:3 molar ratio of trypsin. Under these conditions, an incubation period of 2 h was sufficient to activate the crude aqueous extracts of pancreas powder. 6 h were necessary to activate purified fractions. Carboxypeptidase A activity was measured against the substrate carbobenzoxyglycyl-L-phenylalanine (Z-Gly-Phe) (ref. 5). 16 mM solutions were incubated at 25 ° for 4, 8 and 12 min. The extent of hydrolysis was estimated with ninhydrin. For the activation of Subunit I I (leading to the endopeptidase), aqueous solutions adjusted to p H 7.0 were incubated at o ° for 1-2 h with a 1:20 or 1:5 ° molar ratio of trypsin. Chymotrypsinogen C was activated by a technique previously described 4. In both cases, the activity against a IO mM acetyl-L-tyrosine ethyl ester (ATEE) solution in a 4 mM Tris buffer containing 30 mM NaC1 was measured by titrimetry e. The specific activities were expressed as the number of enzyme units (/zmoles substrate hydrolyzed per min) per mg protein (Ei~m ~ 19 for procarboxypeptidase 5 and 23.8 for chymotrypsinogen C (ref. 3)).

Preparation of procarboxypeptidase A-S6 A modification of the method of YAMASAKI et al. 7 was used. An acetone powder (450 g) was prepared 5 from fresh bovine pancreas (2.2 kg) and stored at --15 °. All subsequent steps were performed at 0-4 ° . The whole procedure must be completed within 8 days. Otherwise, considerable activation of Subunit I I m a y occur. Lots of IO g of the powder were stirred for 2 h at o ° in IOO ml of water. A clear supernatant (80 nil) was obtained by a 45-rain centrifugation at 25 ooo rev./min in the No. 30 rotor of a Spinco Model L centrifuge and was immediately placed on a DEAE-cellulose column (2. 4 cm × 51 cm) equilibrated with a 9 ° mM phosphate buffer (pH 7.0). The column was washed with the buffer, and the procarboxypeptidase was eluted with a 9 ° to 30o mM linear phosphate gradient at the sartle pH. The fractions displaying both Z - G l y - P h e and A T E E potential activity were immediately frozen at --15 ° in order to prevent activation. As shown b y Fig. ia, the procarboxyBiochim. Biophys. Acta, 181 (1969) 82--92

84

R.J. PEANASKY ¢t al.

peptidase peak began to emerge at about 0.2 M phosphate. Direct activity against Z-Gly-Phe and A T E E in the fractions was not higher than 4% and O.l-O.5% of the potential values, respectively. The yield, calculated from the total number of potential units introduced into the column was 80% for carboxypeptidase and 22% for the endopeptidase. This latter value is in agreement with previous estimations s according to which 70-80% of the total potential activity of bovine pancreas against ATEE corresponds to chymotrypsinogens A and B.

a

......

. .G3.1{ 0.24%

b

o,14#

""

.......

.--'"

,-" 0.31 Q2t "~

o.1-1~_

o_

7

£ U

-5

20

40

60

61f~X

Fraction20 No. 40

Fig. i. Purification of bovine p r o c a r b o x y p e p t i d a s e A b y c h r o m a t o g r a p h y on DEAE-cellulose. a. F i r s t c h r o m a t o g r a p h y of t h e e x t r a c t s on a 2. 4 c m X 51 c m c o l u m n ; fraction volume, 14.8 ml; flow rate, 60 ml/h. T h e a b s o r b a n c e of t h e f r a c t i o n s a t 280 mff ( - - - - - - ) a n d t h e i r potential a c t i v i t y a g a i n s t Z - G l y - P h e ( O - - O ) or A T E E (O---O) are g i v e n in % of t h e total a m o u n t s i n t r o d u c e d into t h e column, b. Second c h r o m a t o g r a p h y following (NHi),SO 4 p r e c i p i t a t i o n of t h e f r a c t i o n s u n d e r t h e m a i n p e a k in Fig. ia. C o l u m n 2. 4 c m X 36 c m ; fraction volume, 13. 4 m l ; flow rate, 65 m l / h . S a m e s y m b o l s as in Fig. Ia. T h e n u m b e r s indicate t h e p o t e n t i a l specific a c t i v i t y of t h e fractions a g a i n s t A T E E (endopeptidase activity).

The absorbance ratio of the fractions under the procarboxypeptidase peak at 280 and 260 m# was o.8-I.1, indicating the presence of a considerable amount of nucleic acid. Fractions 46-60 in Fig. Ia were pooled and precipitated by the addition of 0.30 g (NH4)2SO 4 per ml. After standing for 3 ° min, the precipitate was collected by centrifugation and dissolved in 7.5 ml of water. At this point, the direct activity was still 4% for carboxypeptidase and O.l-O.5% for the endopeptidase. Diisopropylfluorophosphate (DFP) was added to a concentration of IO raM, and the pH was adjusted to 7.5. After 3 ° rain, no activity against ATEE could be detected. In one experiment, I,IO-phenanthroline 9 was added in order to chelate the zinc atom controlling carboxypeptidase activity. This preparation is later designated as "Zn-free procarboxypeptidase". The solution was dialyzed overnight against a 9 ° mM phosphate buffer (pH 7.0) and submitted to a second chromatography on DEAE-cellulose. Fig. ib shows that a single peak was obtained. Fractions with a potential specific ATEE activity of 75-85 were pooled and immediately frozen to -- 15 °. These preparations were found to be essentially homogeneous by filtration through a 1.4 cm x 200 cm Sephadex G-Ioo column and by disc electrophoresis at pH 8.6. After concentration by (NH4),SO 4 precipitation and dissolution of the precipitate in a few ml of water, their direct activity against A T E E was not higher than 0.5°/0 of the potential value. Biochim. Biophys. Acta, 181 (1969) 82-92

PROCARBOXYPEPTIDASE A AND CHYMOTRYPSINOGEN C

85

Preparation of porcine chymotrypsinogen C This zymogen was purified 4 from porcine pancreatic juice by three chromatographic steps on DEAE-cellulose, Sephadex G-Ioo and CM-Sephadex. The preparations had a potential specific ATEE activity of 15o and no direct activity against this substrate. They were found to be homogeneous by disc electrophoresis at pH 8.6 and starch-gel electrophoresis at pH 8.2. Enzymatic hydrolysis Digestion of performic acid-oxidized chains A by subtilopeptidase A (EC 3.4.4.16) and by carboxypeptidases A and B (EC 3.4.2.1 and 3.4.2.2) was carried out in the usual way 6. The peptides arising from digestion by subtilopeptidase were fractionated by electrophoresis on Whatman 3 MM paper (50 V/cm; pyridine-acetate buffer (pH 3.6) 4 ° mM in pyridine; 9 ° rain) and chromatography (butanol-pyridineacetic acid-water (15 :IO :3:12, by vol.) or butanol-acetic acid-water (4:1:5, by vol.)). The amino acids released by the carboxypeptidases were identified by thin-layer chromatography on the same Silica gel plates as for the thiohydantoins (see below). The solvents were either phenol-water (3:1, by vol.) or methanol-water-I 5 M ammonia (3:1:1, by vol.) (ref. IO). Edman degradation Degradations were performed according to BLOMBACKet al. 11with the following modifications: (a) A solution of the peptides (O.l-O. 5/tmole) in 0. 5 ml water was adjusted to pH 9.0 with lO% N-ethylmorpholine, mixed with 0.5 ml of a N-ethylmorpholine (60 ml)-acetic acid (1.5 ml)-ethanol (500 ml) buffer and condensed with redistilled phenylisothiocyanate (50/,1). (b) The washing of the phenylthiocarbamyl peptides with ethyl acetate was omitted. (c) Cyclization to 2,5-thiazolinone by trifluoroacetic acid was repeated twicO 2 in order to increase the yield which was sometimes low, especially with N-terminal glycine. When the presence of a N-terminal glutamine was suspected, condensation with phenylisothiocyanate was performed immediately and cyclization was carried out only once to avoid the formation of pyrrolidone carboxylic acid derivatives. All the solvents were carefully freed of aldehydes (negative Tollens test) and the reactions were performed under pure nitrogen. The phenylthiohydantoins finally obtained by isomerization of the 2,5thiazolinones were identified by thin-layer chromatography on 2.5 cm × 8 cm plates prepared by immersion in a suspension of 250 g Silica gel in 600 ml chloroformmethanol (I :i, by vol.). When formamide was used, this compound was directly incorporated into the suspension. The plates were developped with Solvents D and E of EDMAN AND SJOQUIST 13 and Solvents IV and V of JEPPSON AND SJOQUIST14. RESULTS

Amino acid sequence of performic acid-oxidized chain A from bovine Subunit I I Subunit II contains an easily recognizable half-cystine residue at the N-terminal position, and it can be activated under mild conditions which induce little activation of Subunit I to carboxypeptidase. Moreover, this latter enzyme, if formed, can be inhibited by the addition of fl-phenylpropionate or by Zn chelation. Therefore, the identification of chain A of Subunit II was attempted directly from the whole actiBiochim. Biophys. Acta, 18I (1969) 82-92

86

R.J. PEANASKY et al.

vated procarboxypeptidase conlplex. When I,Io-phenanthroline and /5-phenylpropionate were used, unwanted degradation by carboxypeptidases A and B was reduced to a minimum, and most of the chain was obtained intact. Degradations were more important when I,IO-phenanthroline was omitted during the purification of procarboxypeptidase. 2.5/,moles of procarboxypeptidase purified in the presence or absence of I,IOphenanthroline were dissolved in 12.7 ml of a o.I M phosphate buffer (pH 7.5) containing o.i M fl-phenylpropionate and the mixture was incubated for 15 min at o ° with trypsin (molar ratio, i :2o). A 2oo-fold molar excess of D F P was added and the pH was readjusted to 7.5. Within 3o min, the endopeptidase activity was completely abolished. The p H was lowered to 5.8 and the mixture was exhaustively dialyzed against water and lyophilized. It was then oxidized by performic acid and lyophilized until free of formic acid. The dry powder was extracted IO times with 5-ml portions of cold water 15. The combined extracts were clarified by passage through a Millipore filter and taken to dryness under vacuum. Results obtained by filtration through Sephadex G-5o of a solution of the dry powder in I ml of 5 mM He1 (Fig. 2) were similar to those already observed with aqueous extracts of performic acid-oxidized bovine and porcine chymotrypsins Aa and B~ (refs. 6, 16, 4). A peak (Peak I) absorbing weakly at 280 m/~ but strongly at 230 m# emerged from the column at the

l/

r o~

t

~

ft\ 7

Retention volume

Fig. 2. Purification of chain A b y Sephadex filtration. The a q u e o u s e x t r a c t s of activated performic acid-oxidized p r o c a r b o x y p e p t i d a s e A were filtered t h r o u g h a I, 4 cm × 200 cm Sephadex G-5o (medium) column equilibrated and eluted w i t h 5 mM HC1. F r a c t i o n volume, 4.2 ml; flow rate, 20 ml/h. Retention volume of the column, 135 ml. The arrow indicates the fractions t h a t were pooled for peptide mapping. Peak I I is fi-phenylpropionate.

beginning of the second retention volume. The fractions under this peak were pooled, concentrated and submitted to peptide mapping by electrophoresis-chromatography on paper. The peptide map obtained was different according to whether i,Io-phenanthroline was present or absent in the course of the purification of procarboxypeptidase. In the presence of this compound, two cysteic acid-containing spots (hatched Spots I and 2 in Fig. 3) were observed. In its absence, Spot 2 was missing and was replaced by a new spot, I', located in the same region as Spot i. The amino acid composition of Spots I, 2 and I ' is given in Table I. Table I shows that a tridecapeptide, a dodecapeptide and a undecapeptide with very similar amino acid composition were obtained. The tridecapeptide arising from Biochim. Biophys. Acta, 181 (1969) 82-92

PROCARBOXYPEPTIDASE A AND CHYMOTRYPSINOGEN C TABLE

87

I

AMINO ACID COMPOSITION OF T H E T H R E E CYSTEIC ACID PEPTIDES FROM ACTIVATED, OXIDIZED PROCARBOXYPEPTIDASE

Numbers in parentheses indicate nearest integers. Aetivated zymogen Amino acid

Zn-free procarboxypeptidase Spot 2 Spot x

Ala Arg Asx* CySOsH Glx** Gly lie Leu Phe Pro Ser Total

2.00 (2) o.81 (i) 1.16 (I) 0.53 (I) 1.3 ° (I) 1.2o (I) I.OO (I) 0.97 (I) 0.78 (I) 1.7o (2) 0.90 (I) 13

Zn-containing procarboxypeptidase Spot z"

1.62 (2) o.17 (o)

i.o8 (i) o.oo (o)

0.98 (I)

0.57 1.19 1.35 0.99 1.o2 o.68 1.99 I.OI 12

I.I 9 (I)

(I) (I)

0.79 ( I ) 1.o8 (I) 1.o4 (I) i.oo (i) 0.97 (I) 0.83 (I) 1.94 (2) 0.98 (I) iI

(I)

(I) (I) (1) (2) (I)

* (Asp + Asn). "* ( G l u + Gln).

Zn-free p r o c a r b o x y p e p t i d a s e c o n t a i n e d a C - t e r m i n a l a r g i n i n e residue a n d it could t h e r e f o r e be a s s u m e d to c o r r e s p o n d to t h e u n d e g r a d e d c h a i n of S u b u n i t I I . I n t h e o t h e r two peptides, a r g i n i n e or a r g i n i n e a n d a l a n i n e were lacking. These two p e p t i d e s were o b v i o u s l y d e r i v e d f r o m t h e first b y a l i m i t e d h y d r o l y s i s b y traces of a c t i v e c a r b o x y p e p t i d a s e A a n d B r e m a i n i n g in t h e i n c u b a t e d m i x t u r e s . T h e s e q u e n c e of t h e t r i d e c a p e p t i d e was w o r k e d o u t as r e p o r t e d i n T a b l e I I . Six successful E d m a n degrad a t i o n s p r o v i d e d a n o v e r l a p b e t w e e n t h e s u b t i l o p e p t i d a s e p e p t i d e s S 1 a n d S 2. T h e n ,

TABLE II SEQUENCE OF T H E PERFORMIC ACID-OXIDIZED T R I D E C A P E P T I D E (SUBUNIT

II)

Sequence No. Peptide

r

Tridecapeptide Dodecapeptide Undecapeptide Edman degradation of the tridecapeptide Digestion of the undecapeptide by subtilopeptidase and Edman degradation of the resulting peptides $1 S,

(CySOsH, Gly, Ala, Pro, Ile, Phe, Glx, Pro, Asx, Leu, Ser, Ala, Arg) (CySOsH, Gly, Ala, Pro Ile, Phe, Glx, Pro, Asx, Leu, Ser, Ala) (CySO3H, Gly, Ala, Pro, Ile, Phe, Glx, Pro, Asx, Leu, Ser)

Ss

Sequence

2

3

4

5

6

7

8

9

Io

x/

z2

z3

CySOsH-Gly-Ala-Pro-Ile-Phe

CySOsH-Gly-Ala-Pro-Ile Phe-Gln-Pro-Asn Leu-Ser CySO3H-Gly-Ala-Pro-Ile-Phe-Gln-Pro-Asn-Leu-Ser_Ala_Arg Biochim. Biophys. Aeta, 181 (1969) 82-92

88

R . J . PEANASKY et

al.

Electro )horesis

,--~ c,'

.,':c9

+

"-~'

@ 1

CO

Fig. 3. Electrophoresis-chromatography of the peptides under Peak I in Fig. 2. This particular experiment was performed with Zn-free proearboxypeptidase. The hatched spots correspond to cysteic acid-containing peptides. See text for experimental conditions.

the peptide Leu-Ser ($3) could be unequivocally located between $2 and the C terminal Ala-Arg sequence. The position of this sequence of 13 residues in the procarboxypeptidase A molecule is undoubtedly at the beginning of the N-terminal sequence of oxidized Subunit n on account of the following results: (a) The insoluble proteins left after aqueous extraction of the peptides contained two of the N-terminal residues (lysine and aspartic acid) normally present in oxidized procarboxypeptidase, but the Nterminal cysteic acid residue was no longer in the protein fraction and was replaced by valine. It is already known that an N-terminal valine residue appears during activation of Subunit n (ref. i). (b) From a subtilopeptidase digest of DNP-oxidized procarboxypeptidase, the peptide DNP CySO3H (Gly, Ala, Pro, Ile) was identified.

Sequence of the performie acid-oxidized chain A from porcine chymotrypsinogen C It was reported in a recent publication 4 that the activation of porcine chymotrypsinogen C involved the cleavage of an arginyl-valine bond situated near the N-terminus of the molecule. After performic acid oxidation, the short chain A was isolated as described above b y Sephadex filtration and electrophoresis-chromatography. It was found to be a tridecapeptide with the following composition : (CySO3H)1, Gly 1, Val 1, Pr%, Ser 2, Phe 1, Asx 1, Leu 1, Ala 1, Arg 1. In subsequent experiments, two other cysteic acid-containing spots were detected after electrophoresis-chromatography. These spots corresponded, respectively, to a decapeptide ((CySO3H)v Gly 1, Val v Pro s, Ser,, Phe 1, Asx 1, LeUl) and to a nonapeptide ((CySOaH)I, Gly 1, Val,, Pro a, Ser D Phe v ASnl). Analysis showed that the decapeptide differed from the tridecapeptide only by the loss of one residue each of serine, alanine and arginine. A leucine residue was also lacking in the nonapeptide. Hence, it could be assumed, as in the case of Subunit II, that the intact peptide contained 13 residues and that it was partly degraded b y enzymatic attack. The tridecapeptide was found to have an N-terminal cysteic acid residue and to release arginine and alanine by digestion b y carboxypeptidases A and B. The nonapeptide was digested with subtilopeptidase and the sequence of the two resulting peptides was ascertained by E d m a n degradation. Evidence provided by these methods is summarized in Table I I I . The peptide S 1, containing an N-terminal cysteic acid, must obviously be put first in the sequence. It is followed by $2, as indicated b y the composition of the Biochim. Biophys. Acta, 181 (1969) 82-92

89

PROCARBOXYPEPTIDASE A AND CHYMOTRYPSINOGENC TABLE III

SEQUENCE OF THE SECONDTRIDECAPEPTIDE (N-TERMINALSEQUENCEOF CHYMOTRY'PSINOGENC) Sequence No. Peptide

I

2

3

4

5

6

7

8

9

~o

~

~2

z3

Tridecapeptide (CySOsH, Gly, Val, Pro, Ser, Phe, Pro, Pro, Asx, Leu, Set, Ala, Arg) (CySOsH, Gly, Val, Pro, Ser, Phe, Pro, Pro, Asx, Leu) Decapeptide Nonapeptide (CySO3H, Gly, Val, Pro, Ser, Phe, Pro, Pro, Asx) Digestion of the nonapeptide by subtilopeptidase and Edman degradation of the resulting peptides CySOsH-Gly-Val-Pro-Ser $1 Phe-Pro-Pro-Asn $2 Digestion of the tridecapeptide with carboxypeptidases A and B Ala-Arg N-terminal residue in tridecapeptide CySOsH Sequence CySOsH-Gly-Val-Pro-Ser-Phe-Pro-Pro-Asn-Leu-Ser-Ala-Arg

nonapeptide, and then by Leu, as indicated by the decapeptide. The second serine residue occupies position II, between the decapeptide and the C-terminal Ala-Arg sequence.

Absence of tryptophan in the N-terminal sequence of chymotrypsinogen C Tryptophan, which is transformed into N-formyl kynurenine by performic acid, might have been present but undetected in the oxidized peptides. Final proof of the absence of this residue required the preparation of the intact reduced chain. In order to avoid any degradation, the experiment was performed with reduced S-carboxymethylated chymotrypsinogen C which was exhaustively digested by trypsin. Among the tryptic peptides separated by Sephadex filtration and eiectrophoresis-chromatography on paper, one had the same composition as the tridecapeptide described before, except for cysteic acid which was replaced by S-carboxymethyl cysteine. This peptide had a low molar extinction coefficient in the 27o-29o-m # region and gave a negative response with the Ehrlich reagent. Thus, it was clear that the intact chain did not contain tryptophan.

DISCUSSION

As expected, the short chain arising during the tryptic activation of Subunit II in bovine procarboxypeptidase A could be fully identified without previous separation of this subunit from the trimer. After activation and performic acid oxidation, three cysteic acid peptides were identified. The largest one, a tridecapeptide, contained a C-terminal arginine and was assumed to correspond to the intact chain. Similar observations were made with activated and performic acid-oxidized chymotrypsinogen C. Moreover, the absence of tryptophan in the N-terminal sequence of chymotrypsinogen C was demonstrated by an independant assay. It was concluded Biochim. Biophys. Acta, I81 (I969) 82-92

R . J . PEANASKY et al.

9°

that the short chain in chymotrypsin C and also in the endopeptidase derived from Subunit II contained 13 residues only. Like the other chymotrypsinogens known so far, bovine Subunit II and porcine chymotrypsinogen C are activated by the cleavage of the first basic bond in the N-terminal sequence of the molecule. This observation is of special interest in the case of Subunit II whose relations to the chymotrypsinogens are now definitely ascertained. The fact that the activating cleavage occurs at the I3th position instead of the I5th as in chymotrypsinogens of the A and B type does not mean that the mode of activation is fundamentally different. It merely reflects a different position of the first basic residue in the sequence. The assumptionS, 4 according to which bovine Subunit II resembles porcine chymotrypsinogen C more than it does the chymotrypsinogens A and B from bovine and porcine pancreas is well supported by the present experiments. The N-terminal sequences of 6 chymotrypsinogens, including chymotrypsinogen C and Submfit II, are listed in Table IV for comparison. Although restricted to 15 residues, this comparison illustrates some points of interest. The fact that the 6 zymogens belong to the same family (the chymotrypsinogen family) is clearly indicated by the existence of 7 invariant residues (Cys (i), Gly (2), Pro (4), Pro (8), Leu (IO), Ser (ii), Arg (15)) and by 2 "conservative" substitutions (VaNAla, Ile Pile at positions 3 and 6). The replacement of isoleucine by another hydrophobic residue (valine) on the right of T A B L E IV N-TERMINAL SEQUENCES IN CHYMOTRYPSINOGENS (ChTg) A, B AND C I n v a r i a n t residues are in italics. [-T-~,activating cleavage by trypsin. Chymotrypsinogen

Sequence No. z

2

3

4

5

6

7

8

9

xo

Ii

12

z3

±4 z5

x6 z7

[] Bovine chymotrypsinogen A (ref. 17)

-Cys-Gly-Val-Pro-Ala-Ile-Gln-Pro-Val-L eu-Ser-Gly-Leu- S er-A rg-Ile-Val

Bovine chymotrypsinogen B (ref. 6)

Cys-Gly-Val-Pro-Ala-Ile-Gln-Pro-Val-Leu-Ser-Gly-Leu-Ala-A rg-I]e-Val

Porcine chymotrypsinogen A (ref. I6)

-Cys-Gly-Val Pro-Ala-Ile-Pro-Pro-Val-Leu Ser-Gly-Leu-Ser-Arg-Ile-Val

Porcine chymotrypsinogen B (ref. 4)

-Cys-Gly-Val-Pro-Ala-Ile-Pro-Pro-Val-Leu-Ser-Gly-Leu-Ser-A rg-Ile-Val

[] []

[] Porcine chymotrypsinogen C

-Cys-Gly-Val-Pro-Ser-Phe-Pro-Pro-Asn-Leu-Ser

Ala-A rg-

[] Bovine Subunit II -Cys-Gly-Ala-Pro-Ile-Phe-Gln-Pro-Asn-Leu-Ser (Bovine chymotrypsinogen C) Biochim. Biophys. Acta, 181 (1969) 8 2 4 2

AIa-A rg-

PROCARBOXYPEPTIDASE A AND CHYMOTRYPSINOGEN C

91

the activating cleavage in Subunit II and chymotrypsinogen C is noteworthy in the light of the role attributed to this residue in the formation of the active site18,19. Moreover, Table IV shows that the two residues missing from the N-terminal sequence of chymotrypsinogen C and Subunit II correspond to deletions at positions 12 and 13. The high degree of homology between the two zymogens is further indicated by the fact that only 3 residues are different in both sequences. Two residues are different in the other group, including bovine and porcine chymotrypsinogens A and B. In contrast, the number of differences increases to 6-8 when a zymogen of one group is compared with a zymogen of the other. The valine residue on the right of the activating cleavage in Subunit II and chymotrypsinogen C is not indicated in Table IV. It may be either at position 16 (substitution Ile-Val) or at position 17 (if an additional deletion occurs at position 16). These results are consistent with the view 4 that at least two chymotrypsinogen lines exist in bovine and porcine pancreas. One provisionally includes chymotrypsinogens A and B. These zymogens are very similar, and they cannot be differentiated 4 before the sequence of the porcine proteins is known. The other line appears to include porcine chymotrypsinogen C and bovine Subunit I I The designation of this latter as bovine chymotrypsinogen C is probably justified.

ACKNOWLEDGMENTS

The authors wish to thank Professor P. Desnuelle for helpful discussions during the completion of this work and the preparation of the manuscript. Financial help was provided by D616gation G6n6rale A la Recherche Scientifique et Technique (Convention No. 66 oo 056). R. J. Peanasky was supported by a U.S. Public Health Service Research Career Program Award 5-K3-4282-o7 and gratefully acknowledges the support of GM0-8825 from the U.S. Public Health Service and the Commonwealth Fund for travel. REFERENCES I J. R. BROWN, R. N. GREENSHIELDS,M. YAMASAKI AND H. NEURATH, Biochemistry, 2 (i 963) 867 . 2 J. R. BROWN, M. YAMASAKI AND H. NEURATH, Biochemistry, 2 (1963) 877. 3 J. E. FOLK AND W. E. SCHIRMER, J. Biol. Chem., 24o (I965) 181. 4 D. GRATECOS, O. GUY, M. :RovERY AND P. DESNUELLE, Biochim. Biophys. Acta, 175 (1969) 82. 5 P. J. KELLER, E. COHEN AND H. NEURATH, J. Biol. Chem., 223 (1956) 457. 6 0 . GuY, D. GRATECOS, M. ROVERY AND P. DESNUELLE, Biochim. Biophys. Acta, 115 (1966) 404 . 7 M. YAMASAKI, J. R. BROWN, D. J. Cox, R. N. GREENSHIELDS, R. W. WADE AND H. NEURATH, Biochemistry, 2 (1963) 859. 8 M. CHARLES,Biochim. Biophys, Acta, 92 (1964) 319. 9 R. PIRAS AND B. L. VALLEE,Biochemistry, 6 (1967) 348. IO M. BRENNER, A. NIEDERWlESER AND G. PATAKI, in E. STAHL, Thin Layer Chromatography, Academic Press, New York, 1965, p. 391. i i B. BLOMBACK, M. BLOMB*CK, P. EDMAN AND B. HESSEL, Biochim. Biophys. Acta, 115 (1966) 371 • 12 P. EDMAN AND G. BEGG, European J. Biochem., i (1967) 8o. 13 P. EDMAN AND J. SJ~QUIST, Acta Chem. Scand., io (1956) 15o7. 14 J. O. JEPPSON AND J. SjoQuIST, Anal. Biochem., 18 (1967) 264. 15 DINH VAN HOANG, M. ROVERY AND P. DESNUELLE,Biochim. Biophys. :4cta, 58 (1962) 613.

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