Isolation and characterization of the cyanogen bromide peptides of 2 forms of porcine pancreatic amylase

BIOCHIMIE, 1971, 53, 957-968.

Isolation and characterization of the cyanogen bromide peptides of 2 forms of porcine pancreatic amylase. P. (',)ZZONE, I,.

PASI~RO,B.

BEAUPOIL and G. MARCHIs-M<)UREN.

l u s t i l u t de C h i m i e Biologique, Universitd de Prooence, P l a c e V.-Ht~go I'~ - M a r s e i l l e (30. (12-5-t971).

S u m m a r y . - - Porcine pancreatic amylases I and II have identical activities, molecular weight, end groups and amino acid composition (except for aspartic acid and/ or asparagine residues). In order to get more information about the structure of the 2 forms, both amylases were cleaved by cyanogen bromide and the fragments thus obtained were purified by gel filtration and analyzed. Kinetics of the cleavage indicates a complete and specific splitting at the methionine level. Successive filtrations of the peptides were performed in 30 p. cent propionic acid on appropriate columns. Nine pure peptides were thus obtained and characterized by their molecular wei,ght, end groups and amino acid composition. Tbe location of the 4 disulfide bridges and of the 2 free SH groups in the peptides was determined. The N and C-terminal peptides were identified, Surprisingly, no difference was detected among the homologous peptides obtained from amylase I and amylase II. This result does not account for the difference of 5 aspartic acid a n d / o r asparagine residues observed at the amylase level. Two hypotheses to interpretate this discrepancy are discussed.

INTt-~OI) UCTION. P o r c i n e p a n c r e a t i c a m y l a s e s 1 a n d II (~.-1,4 giuc a n 4 - g l u c a n o h y d r o l a s e , EC 3.2.1.1.) h a v e b e e n p r e v i o u s l y p u r i f i e d and s h o w n to be e q u a l l y a c t i v e [1-2]. In v i e w of a b e t t e r u n d e r s t a n d i n g of the r e l a t i o n b e t w e e n a m y l a s e s t r u c t u r e and function, and also of this h e t e r o g e n e i t y , c o m p a r a t i v e s t r u c t u r a l studies w e r e p e r f o r m e d in our l a b o r a tory. M o l e c u l a r w e i g h t , end g r o u p s and a m i n o a c i d c o m p o s i t i o n of b o t h m o l e c u l a r f o r m s h a v e been a l r e a d y r e p o r t e d [3!. T h e s e results i n d i c a t e d that a m y l a s e s consist of a single p o l y p e p t i d e c h a i n of about 475 residues. At this stage, the o n l y d e t e c t a b l e d i t r e r e n c e w a s that a m y l a s e 1 c o n t a i n s 5 m o r e a s p a r t i c acid a n d / o r a s p a r a g i n e r e s i d u e s t h a n a m y l a s e II. More p r e c i s e c o m p a r i s o n had t h e n to be r e a c h e d by s t r u c t u r a l studies of fragm e n t s m o r e easily a c c e s s i b l e to c h e m i c a l analysis. C o n s i d e r i n g the size of the a m y l a s e m o l e c u l e and in o r d e r to o b t a i n a l i m i t e d n u m b e r of p e p t i d e s , c h e m i c a l c l e a v a g e of the c h a i n by c y a n o g e n brom i d e was c a r r i e d out. As s h o w n by G n o s s a n d WITxOU [4] s p e c i f i c splitting o c c u r s at the nlethion i n e r e s i d u e s . S i n c e 8 m e t h i o n i u e s are p r e s e n t , 9 p e p t i d e s w e r e then e x p e c t e d . T h e p r e s e n t Abbreviations : T.F.A. : Trifluoroacetic acid. CNBr : Cyanogen bromide. E.D.T.A. : ethylenediamine tetraacetic acid. Dansyl chloride : 1-dimethyl aminonaphtalene-5-sulfonyl chloride.

r e p o r t (*) d e s c r i b e s the o b t e n t i o n , i s o l a t i o n and c h e m i c a l c h a r a c t e r i z a t i o n of the n i n e f r a g m e n t s r e s u l t i n g f r o m s e p a r a t e CNBr c l e a v a g e s of a m y lases I a n d II. F u r t h e r c o n t r i b u t i o n to the e l u c i d a t i o n of the a m y l a s e p r i m a r y s t r u c t u r e by o r d e r i n g the p e p tides in the p r o p e r s e q u e n c e has also b e e n c a r r i e d out u s i n g an i s o t o p i c t e c h n i q u e ; this w o r k w i l l be r e p o r t e d in a f o r t h c o m i n g p a p e r . MATERIALS. H e m o g l o b i n and c y t o c h r o m e C ( h o r s e heart) were purchased from Koch-IAghi Laboratories (England). C a r b o x y p e p t i d a s e s A a n d B and t r y p s i n were from Worthington Biochemical Corporation (U.S.A.). I n s u l i n w a s f r o m S i g m a C h e m i c a l Comp a n y (U.S.A.). T r i f l u o r o a c e t i c acid, c y a n o g e n bromide, ethylene imine, iodoacetic acid (recrystallized b e f o r e use) and 1 - d i m e t h y l a m i n o n a p h t a l e n e 5-sulfonyl c h l o r i d e w e r e o b t a i n e d f r o m F l u k a A.G. ( S w i t z e r l a n d L P u r e p r o p i o n i c a c i d ( P r o l a b o F r a n c e ) w a s r e d i s t i l l e d , if not, a slight p r e c i p i tate m i g h t o c c u r u p o n d i l u t i o n w i t h d e i o n i z e d w a t e r . All S e p h a d e x Gels and Blue D e x t r a n 2000 were from Pharmacia (Sweden). 2,4-dinitrophe(*) Preliminary reports of this work were given at the FEBS congress in Madrid (1969) and at the International congress of Biochemistry in Interlaken (1970).

P. Cozzone, L. Pasdro, B. Beaupoil and G. Marchis-Mouren.

958

uylalanine, used as m a r k e r for gel filtration and all dansyl am i n o acids w e r e from British Drug Houses (England). Silicagel was p u r c h a s e d from Merck A.G. (Germany), T r y p t o p h a n was from S c h w a r z Bioresearch (U.S.A.), norleucine, cysteic acid and S - c a r b o x y m e t h y l c y s t e i n e as standards for amino acid analysis w e r e from Calbiochem (Switzerland). Hydrogen p e r o x i d e and f o r m i c acid for oxidation w e r e from Prolabo (France). All solvents w e r e freshly distilled before use. All other ch em ic a l s w e r e of analytical grade.

METHODS.

Preparation of porcine pancreatic amylases. Amylase was purified from p o r c i n e p a n c r e a s tissue homogenate a c c o r d i n g to FISCHER and B~RNFEI.D I51. Amylases l and II w e r e separated by c h r o m a t o g r a p h y on DEAE-celhdose as previously described !11. The purity of the preparations was routinely c h e c k e d by p o l y a c r y l a m i d e disc el ect r o p h o r e s i s at pH 8.6 and by a m in o acid analysis [2].

Assay procedures. Protein was d e t e r m i n e d by the method of LOWRY calibrated w i t h crystalline serum albnmin. The amylase content of pure p r e p a r a t i o n was measured s p e c t r o p h o t o m e t r i c a l l y (E ~ p1 cm - ~ at 280 nm = 25) and by amino acid analysis. Amylase activity was measured by r e d n c t o m e t r y w i t h d i n i t r o s a l i c y l i c acid as described by NOEI,TING I71.

Cyanogen bromide cleavage. Lyophilised amylase was dissolved in 70 p. cent TFA to a final c o n c e n t r a t i o n of 1 p. cent ( w / v ) and 1.2 fold excess ( w / w ) of cyanogen b r o m i d e was added. The reaction was allowed to p r o c e e d for 28 h at room temperature. Upon completion of the reaction, a 10 fold excess of cold w at er was added. Excess reagents and volatile p r o d u c t s were then r em o v e d e i t h e r by lyophilisation or c o n c e n t r a t i o n in a rotary evaporator. When c h e c k i n g the extent of the reaction, as a function of time, a p p r o p r i a t e aliquots of the m i x t u r e w e r e taken off and treated as described above.

Gel filtration. All filtrations were c a r r i e d out in 30 p. cent p r o p i o n i c acid. The Sephadex columns w e r e previously equilibrated for at least two days w i t h

BIOCHIMIE,

1 9 7 1 , 53, n ° 9.

the same solvent. The t e m p e r a t u r e of the columns was set at 4°C to avoid possible alteration of the p o l y d e x t r a n gel. U n d e r these conditions, the gel a p p e a r e d to be stable for at least six months since the p a c k i n g of the column and the resolution r e m a i n e d unchanged. The effluent was continuously m o n i t o r e d at 280 nm using an < r e c o r d e r (Seive - Paris - France). Because of the high absorption of p r o p i o n i c acid, no rec o r d i n g at 230 nm was possible. F r a c t i o n aliquots (100 vd) w e r e reacted w i t h n i n h y d r i n after alkaline hydrolysis [8!.

Preparation of the samples. The lyophilized CNBr reaction m i x t u r e (300 my) was dissolved in 30 p. cent p r o p i o n i c acid at the c o n c e n t r a t i o n of 10-15 m g / m l . A few drops of c o n c e n t r a t e d p r o p i o n i c acid w e r e occasional)' added to achieve complete solubilization. To l o w er aggregate formation, the samples w e r e incubated for 15 hours at r o o m temperature. A p p r o p r i a t e fractions from gel filtrations w e r e pooled and c o n c e n t r a t e d by r o t a r y ev ap o r at i o n at 3 0 ° C u n d er r ed u ced pressure to obtain a 1015 m g / m l final c o n c e n t r a t i o n in 30 p. cent propionic acid.

Nomenclature. Pooled p e p t i d e - c o n t a i n i n g fractious w e r e named a c c o r d i n g to their position in the elution pattern using capital letters in the alphabetic order. Purified peptides w e r e numbered, a c c o r d i n g to their position in the elution patterns, using roman numbers from I to IX.

Molecular weight estimation. Molecular weights of the CNBr-peptides w e r e estimated by gel filtration on Sephadex a c c o r d i n g to the general metbod described by ANDREWS [9], except that 30 p. cent p r o p i o n i c acid was used as eluting solvent. A sephadex G-100 (100 × 2.5 cm) column was used for peptides I and H, and a Sephadex G-75 (100 × 2.5 era) column for peptides III, V and VI. Molecular weights of the other peptides w e r e estimated either by filtration on G-50 (10'0 × 2.5 era) column or G-25 (100 × 1.5 era) column. F o r calibration, t r y p si n (M.W. = 23,800), bovine h e m o g l o b i n covalent subunit (M.W = 17,000), horse heart c y t o c h r o m e C (M.W. = 12,400), insulin (M.W. = 5,700) and DNP-alanine (M.W. = 255) w e r e used as markers. The column void volumes (Vo) w e r e d e t e r m i n e d by using Blue Dextran 2000. F o r each peptide or protein, the elution volume (Ve) was measured at the maxim u m of the peak plus one half of the sample volume.

Cyanogen bromide peptides of pancreatic amylase. Oxidation and S-carboxymethylation. Each peptide was oxidized with performic acid a c c o r d i n g to Hirs' procedure [10J. At the end of the reaction h y d r o b r o m i c acid was added to destroy excess performic acid. The peptides were recovered from volatile products by lyophilisation. In an alternate e x p e r i m e n t S-carboxymethylation was used to modify the half-cystinyl residue. Each purified peptide w a s reduced by ~-mercaptoethanol in 8 M urea a n d tris buffer (pH 8.6) and then alkylated by iodoacetic acid II1~. Desalting of the larger S-carboxymethylated peptides was achieved by dialysis where-as smaller peptides were directly applied on either Sephadex G-75, G-50 or G-25 colums.

Amino acid analysis. Peptides (about 0.05 to 0.1 ~M) were hydrolyzed in tetradistilled 5.7 N HC1 in evacuated, sealed tubes at 110°C for 24 h a c c o r d i n g to the method of MOORE and STEIN [12]. H y d r o c h l o r i c acid contained 20 ~tg of phenol per ml. The a m i n o acid composition of the resulting hydrolyzate was d e t e r m i n e d on a ¢ T e c h n i c o n autoanalyzer >>. Correction factors were applied for the loss of t h r e o n i n e and serine d u r i n g hydrolysis. The halfcystine content was measured as cysteic acid following performic oxidation or as S-carboxymethylcysteine after reduction and alkylation of sulfhydryl groups. The integration constants for homoserine and its lactone were d e t e r m i n e d on purified samples. In agreement w i t h earlier publications [13] when the factors for leucine and lysine were 47.5 and 49, the following values were used : homoserine : 41, lactone : 29. The procedure of AMBLER [14] tO convert all homoserine lactone into homoserine, was not routinely employed since the peak of lactone was still present on the chromatogram, p r o b a b l y due to some isomerisation on the column. Moreover, the treatment with p y r i d i n e acetate buffer pH 6.5 at 105°C i n d u c e d some degradation in the hydrolyzate, namely the glutamic acid content was about 20 p. cent lower than expected. The separation of amino acids was performed in a single 0.62 × 65 cm column packed with C-2 chromobeads. Tile usual gradient was modified and an excellent separation of all the amino acids i n c l u d i n g homoserine and its lactone was reached w h e n filling each of the nine cascade chambers of the autograd as follows :

Chamber 1 38 ml 0.2 N sodium citrate buffer pH 2.75 c o n t a i n i n g l0 p. cent methanol + 2 ml methanol. BIOCHIMIE, 1971, 53, n ° 9.

Chamber 2

959

12 ml 0.2 N sodium citrate buffer

pH 2.75. 28 ml 0.2 N sodium citrate buffer pH 2.88.

Chambers 3 40 ml 0.2 N sodium citrate pH 2,88 4-5 Chambers 6 40 ml 1.4 N sodium citrate pH 6.10. to 9 All the buffers, except the pH 6.10 buffer, contained thiodiglycol (5 ml per liter). The column was previously equilibrated in 0.2 N citrate buffer pH 3.0, 10 p. cent in methanol and c o n t a i n i n g 1 p. mille EDTA (w/v). The entire separation time was 5.30 h at 60°C, and the flow rate 0.83 m l / m n .

Tryptophan analysis. The t r y p t o p h a n content of each peptide was deter miRed before h y d r o l y s i s by the colorimetric technique of SPIES and CHAMBERS [15]. To avoid i n t e r f e r i n g coloration p r o b a b l y due to m i n o r amounts of solubilized p o l y d e x t r a n material, an ultimate filtration on Sephadex G-10 (10 X 0.6 cm) column in 30 p. cent p r o p i o n i c acid was performed. The amount of t r y p t o p h a n was also d e t e r m i n e d spectrophotomelrically by calculating the t r y p t o p h a n to tyrosine ratio according to the method of GOODWIN and MOUTON [16!.

End group analysis. For the d e t e r m i n a t i o n of the C-terminal residues, the digestion of peptides by carboxypeptidase A was carried out according to the method described by BROWN [17]. Commercial DFPtreated carboxypeptidase A was treated again by DFP just before use according to the procedure [ of FRAENKF~L-CoNRATet al. [181. The enzyme-tosubstrate molar ratio was about 1 to 20. The free a m i n o acids released by the digestion were characterized on a ¢ T e c h n i c o n autoanalyzer ~. Two techniques were employed for the characterization of a m i n o t e r m i n a l residues by dansylation, d e p e n d i n g on the size and soluoility of the different peptides. The method of GRos and LABOUESSE [191 was applied to larger peptides namely peptides I, II, III, V and VI. For smaller peptides, i.e. peptides-IV, VII, VIII and IX, the c o n d e n s a t i o n reaction was performed using a more simple procedure according to TRUFANOV [20]. In both cases, the identification of dansyla m i n o acids was achieved by a t w o - d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a p h y on 20 cm × 20 cm silicagel plates, 250 ~ thick, successively in sol65

P. Cozzone, L. Pasdro, B. B e a u p o i l a n d G. M a r c h i s ~ V l o u r e n .

q60

vent systems II and IV of Gnos and LABOUESSEfor the aqueous phase, and in solvents I and II for the ether phase. RESULTS.

Kinetics o[ amylase cleavaqe by c!lanogen brom ide. The time course of the reaction of amylase w i t h cyanogen b r o m i d e u n d e r the c o n d i t i o n s d e s cr i b ed in <(Methods>> is given in Fig. 1. Respective amounts of residual m e t h i o n in e , of h o m o s e r i n e and its lactone w e r e d e t e r m i n e d in acid hydrolyzates after various reaction times with cyanogen bromide. T h e reaction p r o c e e d e d r a p i d l y in the b eg i n n i n g then slowed down, and 24 to 28 hours at 25°C w e r e necessary to r e a c h completion. The reaction was stopped after 28 hours when a 95-100 p. cent loss of m e t h i o n i n e was obtained. As shown in Fig. 1 at any one time, the sum (in mole) of methionine, h o m o s e r i n e and its lactone is equal to the n u m b e r of moles of met h i o n i n e per mole of amylase. T h e f o r m a t io n of m e t h i o n i n e sulfone and m e t h i o n i n e sulfoxide was found not to o c c u r u n d e r our conditions. In control e x p e r i m e n t s , amylase was incubated with 70 p. cent T.F.A. alone for 28 hours, the incubate was lyophilized or evaporated u n d e r reduced pressure and finally analyzed by polya c r y l a m i d e gel e le c t r o p h o r e s i s : no change in the amylase pattern was found.

S-carboxymethylated, reduced and S-aminoethylated, or oxidized are very poorly soluble in w a t e r and classical buffers. The unmodified peptides w e r e found soluble either in 8 M urea, at pH 5 in p y r i d i n e acetic acid buffer or in c o n c e n t r a t e d organic acid solutions. Separation of the peptides by ion exchange c h r o m a t o g r a p h y in 8 M urea was unsuccessful, probably because each p ep t i d e is present, at least, in two differently ch ar g ed and interconvertible forms, due to the h o m o s e r i n e - - h o m o s e r i n e lactone isomerisation at the C-terminal position ; also heterogeneous charge modifications due to some d eam i d at i o n d u r i n g the a c i d i c t r eat m en t is likely to occur. The separation was then a c h i e v e d by m o l ecu l ar sieving on p o l y d e x t r a n gels. 50 p. cent acetic acid p r e v i o u s l y used as a solvent [21] was ab an d o n ed because of its c h e m i c a l reactivity w i t h respect to the peptides and the p o l y d e x t r a n material [22]. Finally, all filtrations w e r e c a r r i e d out in m o r e suitable 30 p. cent p r o p i o n i c acid. FHACTbO~JS t

I

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FI6. 2. Elution profiles of the separation of the peptides, obtained after cleaving amylase by cyanogen bromide. The column system was composed of two (2.5 × 100 cm) columns of Sephadex G-100 connected in series. The sample (300 mg) was applied and the column developed by ascending elution. Solvent : 30 p. cent propionic aci d ; temperature: 4°C: flow r a t e : 15 ml/h. Void volume : 275 ml. Tube-fraction volume: 5 ml ( - - ) : Absorbance at 280 rim; ( ......... ) : Ninhydrin profile. Fractions were pooled as indicated by the solid bars. -

i

[

I

io Flfi. 1. - - Time course of conversion of methionine residues in porcine pancretic amylase ~vith cyanogen bromide in 70 p. cent trifluoroacetic acid. Solid thin vertical line, unreacted methionine ; broken line, homoserine ; solid mide line, homoserine lactone.

Kinetics of cleavage and separation of CNBr-peptides w e r e separately p e r f o r m e d amylases I and II. The rate and the extent m e t h i o n i n e c o n v e r s i o n was found i d e n t ic a l both enzymes.

the on of for

Separation and characterization of Peptides. The peptides in the whole cleavage m i x t u r e either modified, r e d u c e d by m e r c a p t o e t h a n o l and

BIOCHIMIE, 1971, 53, n ° 9.

-

The w h o l e cleavage p r o d u c t was first fractionated on Sephadex G-100. I n d e n t i c a l patterns w e r e obtained for both amylases. E l e v e n p e p t i d i c fractions of the eluate n am ed A-H, M1 and M 2 w e r e obtained by p o o l i n g the c o r r e s p o n d i n g tube fractions (Fig. 2). The peptides co n t ai n ed in these fractions w e r e purified by f u r t h e r gel filtrations on a p p r o p r i a t e p o l y d e x t r a n gels. All these operations are s u m m a r i z e d in Table I. The 9 peptides isolated f r o m these fractions w e r e c o n s i d e r e d as pure w h e n fulfilling all the following h o m o g e n e i t y criteria : 1 °) A s y m m e t r i c a l elution peak by gel filtration, 2 °) Relative elution constant R~ =

C y a n o g e n b r o m i d e p e p t i d e s of p a n c r e a t i c a m y l a s e . Ve/Vo of the peak on a given gel, 3 ° ) Only one C-terminal residue, namely h o m o s e r i n e (except for the C-terminal peptide), 4 °) Only one N-terminal residue (except for the N-blocked peptide), 5 ° ) the n u m b e r of each residue in the amino acid c o m p o s i t i o n being an integer (within 4 p. cent) w h e n using h o m o s e r i n e + hoinoserine lactone : 1 (except for the C-terminal peptide). Moreover, the m o le c u la r w e i g h t thus calculated from the amino acid c o m p o s it io n should equal the one estimated by gel filtration.

Fraction M r - - This is the b r e a k t h r o u g h peak (Fig. 2). The fraction M1 was analyzed for its

961

residue and h o m o s e r i n e at the c a r b o x y l t e r m i n a l end (Table II). Since its amino acid composition (Table III) shows 4 half-cystine residues, thus i n d i c a t i n g possible i n t e r c h a i n disulfide bridges, fraction A 2 was either fully reduced, then S-carh o x y m e t h y l a t e d or oxidized by p e r f o r m i c acid, then applied to the same column of Sephadex G-100. The oxidized or S-alkylated peptide elutes as a single peak at the same position as before c h e m i c a l modification, w h i c h is consistent w i t h a single chain peptide. The m o l ecu l ar weight estimated by gel filtration (Table II) and the one c o m p u t e d from am i n o acid analysis (Table III) are in good agreement. The p ep t i d e in fraction A:~

TABLEAU

I.

Diagram of separation o[ the 9 peptides released after cleavage by CNBr of porcine pancreatic amylase. [

PORCINEPANCREATIC AMYLASE • CNBr

I

~,oo 3o~ PROP,ON,O AC,D

I

I

I

I

I

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[

I

I

I

I

I

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A

B

C

D

E

F

G

H

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A, A= A= B, B 2 B = C~ C ~ C = .

.

.

.

.

.

;

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D~ D=

..k,

E, E=E3 F, F, F3 G,G,G3 H,H, H3- - - ~

L___.~._~

r,. . . .

~

¢-. . . .

oxidation: i

] H3~, D,=

"g'"g""

(1)

@ @ @ ®

@

~

(~

degradation products

Quantitative recovery of the p~ptides was obtained after filtration. Each peptide was present in stoichiometric molar amount with respect to amylase. amino acid content and was found to contain no methionine. The search for aggregates was c a r r i e d out by refiltration on Sephadex G-100 after c o n c e n t r a t i o n and incubation o v e r n i gh t at 25°C. The elution pattern thus obtained is quite s i m i l a r to the one obtained with the total m i x t u r e (Fig. 2). These 2 results are i n d i c a t i v e of aggregated but not uncleaved material.

Fraction A. - - This fraction was r e c h r o m a t o g rap h ed on S e p h a d e x G-200 to give 3 peaks called A1, A2 and A a in their elution order. F r a c t i o n A 1 co n t ai n ed aggregated material similar to M 1 and was discarded. F r a c t i o n A z was further purified on Sephadex G-100 to give pure peptide I (Ve/Vo : 1.35) with valine as amino t e r m i n a l BIOCHIMIE, 1971, 53, n ° 9.

has valine and h o m o s e r i n e as am i n o and carboxyl t e r m i n a l residues, but its amino acid composition is quite different frmn that of the peptide in fraction A2, and was f u r t h er found identical to the one in fraction B2. Fraction B. - - When submitted to gel filtration on Sephadex G-100, fraction B was resolved into 3 peaks B1, B2 and B3 (Fig. 3). The p ep t i d e in fraction B 1 .was r ead i l y identified by its position in the elution d i a g r a m (Ve/Vo : 1.35) and its amino acid composition and m o l ecu l ar w e i g h t with peptide I. The major constituent in fraction B2 is thus c o n t a m i n a t e d by p ep t i d e I. A second refiltration of fraction B e on Sephadex G-100 finally yields pure peptide II, This 85 residues-

P. Cozzone, L. Pasdro, B. Beaupoil and G. Marchis-Mouren.

962

Fraction C. - - Gel f i l t r a t i o n o n S e p h a d e x G-100 o f f r a c t i o n C (Fig. 3) g a v e 3 p e a k s C1, C,., a n d C 3. T h e p e p t i d e i n f r a c t i o n C 1 ( V e / V o = 1.60) w a s i d e n t i f i e d b y i t s e n d g r o u p s a n d a m i n o a c i d c o m p o s i t i o n w i t h p e p t i d e II. T h e a n a l y s i s

l o n g p e p t i d e ( T a b l e s II a n d III) c o n l a i n s a s i n g l e h a l f - c y s t i n e r e s i d u e w h i c h c o r r e s p o n d s to o n e o f t h e 2 f r e e s u l f h y d r y l g r o u p s p r e s e n t in t h e w h o l e a m y l a s e m o l e c u l e [23]. T h e c o m p o n e n t i n f r a c t i o n B 3 w a s f o u n d s i m i l a r to t h e o n e in f r a c t i o n Co.

TABLE II.

Molecular weight and amino and carboxyI terminal residues o[ the 9 CNBr-peptides. Molecular w e i g h t of t h e p e p t i d e s w a s e s t i m a t e d by pionic acid. The m o l e c u l a r w e i g h t of a m y l a s e w a s Amino terminal residues were characterized by their a f t e r c a r b o x y p e p t i d a s e digestion. The N-acetyl group t r o s c o p y (24).

gel filtration, on S e p h a d e x c o l u m n , in 30 p. cent prop r e v i o u s l y d e t e r m i n e d by f o u r different m e t h o d s (3). d a n s y l d e r i v a t i v e s and c a r b o x y l t e r m i n a l r e s i d u e s was d e t e r m i n e d by Nuclear Magnetic R e s o n a n c e specCNBr peptides Amylase

_!

N-terminal

+

i

Val

i --Va,

HSer

~ HSer

residue

C-terminal residue

[1

lII

IV

V

I

VI

i VII

VII'

'

-[I -" i: . . . . . . . . .! 13 500] 9 00n 4 200 3 0001 5 300 4 200 4 0001 3 000 1 2001 + 1 000]-4- 400 ~ 300'-~- 500 + 400 -+- 4001 -I- 300

Molecular weight

(gel filtration)

~

-Lea

, Val

Ser

Leu ~ H S e r

HSer

-Pro HSer

700

--Val N - A e e t y , block ed HSer I

HSer

IX -

+2 300

53 000

Leu

N-Acetyl blocked

HSer

Leu

TABLE III.

Amino acid composilion of the CNBr-peptides of porcine pancreatic amylase and corresponding molecular weight rI

CNBr peptides

A mino acids I

II

X

j 111

r

IV

--

1/2 Cys . . . . . . . . . . . . . . . .

!

V

[

.......

Vl

! Vll

VllI!

IX

Sum oI peptides

Amylase

lO 61-62 (') 66-67 (") 23 32 0 38 21 52 30 37

4

1

1

1

2

1

0

O

0

10

Asx . . . . . . . . . . . . . . . . .

17

14

6

4

10

4

5

3

1

64

Thr ................ Ser . . . . . . . . . . . . . . . . HSer .............. Glx . . . . . . . . . . . . . . . . Pro Gly . . . . . . . . . . . . . . . . Ala . . . . . . . . . . . . . . . . . Vai . . . . . . . . . . . . . . . . . Met . . . . . . . . . . . . . . . . Ile . . . . . . . . . . . . . . . . . Leu . . . . . . . . . . . . . . . . Tyr . . . . . . . . . . . . . . . . Phe . . . . . . . . . . . . . . . . Lys . . . . . . . . . . . . . . . . His . . . . . . . . . . . . . . . . . Arg ................. Try . . . . . . . . . . . . . . . .

6-7

6 7

2 3 0 2

2 1 1 1

1 5 1 3

2 2

2 4

1 1

1 1

23224

1

1

3

4 4 3 0 3 2

2 2 3 0 2 2

7 4 2 0 1 1

1

2

1

2 2

1 1

3 3

1

0

1

1

3

i

8 1

11-12 7 14 8 12 0 6 I 8 6 , 6 : 5 2 8 3

Total . . . . . . . . . . . . . . . . . . . Molecular weight . . . . . . . .

132-134 i

14 879 -15 103

(*) a m y l a s e I ; ( ' ' ) a m y l a s e II.

BIOCHIMIE, 1971, 53, n ° 9.

1

9 2 10 5 6 0 5 3 2 3 3

85

I

1

1

1

2 2 3 1 4 0

2

1

1

2

2 2 3 2

1 1 2 2

1

1

2

1

1

1

2

4

1

1

2 0 2 1

1 0 0 2

i

:

1

1

1

I

1

0

2 1 i 2 2

i

40

28

52

l

40

35

26

4 441

3 881

1

5 2

i

1

2 2 6 3 3 0

12

9 443]I 4 433 3 201 5

682 ,

3 189

2

1

F i

i

2 1 1 1 5 26 3 297

8

34-35 20 52 29 36 0 22 22 18 24 2O 9 25 16 464-466

8

21 23 18 23 2O 9 27 15-17 473-476

Cyanogen bromide peptides of pancreatic amylase. by dansylation of the p e p ti d e in fraction C,, indicates 2 am i n o t e r m i n a l residues (Leucine and Valine) in equal m o la r amounts. Possible contam i n a t i o n by p ep ti d e II w h i c h also possess a valine residue at the amino t e r m i n a l position w a s then e x a m i n e d . Even after an extra filtration on Sephadex G-100, the same 2 t e r m i n a l residues w e r e

k.~ I B2 !

I Ba I

Fraction B ~'~

-0. I I

.1.0

I C~

.

~

r

C2

I

I

iC3

I

i FractionC

~

-0.5

I

I

I

tion of this p ep t i d e at the C-terminal end of the whole amylase molecule. The lack of h i st i d i n e in peptide IV should be noticed. As expected, one half-cystine residue w as found in both peptides. Moreover, amino acid analysis of fraction C 2 as well as the equal amounts of the N-terminal residues valine and leucine can be acco u n t ed for by an e q u i m o l a r m i x t u r e of these 2 peptides. The last peak (Fraction Ca) elutes at 2.26 Vo from the Sephadex (3-100 column. This fraction was found identical to fraction D 1. Fraction D. - - The elution profile obtained w h e n submitting this fraction to gel filtration on Sephadex G-75 consisted of 2 peaks D 1 and D 2 (Fig. 4). The peak D], although almost symmetrical, w as shown to be h et er o g en eo u s by its am i n o acid analysis as well as by end group d e t e r m i n a tion (three N-terminal residues, l eu ci n e valine and serine w e r e found). F i l t r a t i o n of D 1 on Sephadex G-100 was thus necessary. T w o components w e r e separated, the first one Dr1 eluted at 2 Vo and was found s i m i l a r to fraction C,,; the second fraction D]2 contains p u r e p e p t i d e V.

FractionC2

Oxidized

_tO I

TM

963

Ii

~"

t

r

-(~5

D~

I

11

D2'

I

l,0

I

IVo

2Vo

3V o

I

ELUTIONVOLUME

Fro. 3. - - Gel filtration of fractions B and C and oxidized fraction C (Co×) on a (2.5 × 100 era) column of Sephadex G-100. The elution conditions are the same as in Fig. 2. Void volume : 150 ml.

IM Q

I

E~

I I E2

II

E3

I

Z Z

2,0

still found in equal m o la r quantities. On the other hand, the amino acid c o m p o s i ti o n of the peptide in fraction C~ w a s satisfactory w h e n assuming h o m o s e r i n e = 1 and h is t id in e = 1 as a basis of calculation. Since the a m i n o acid analysis indicated the presence of 2 half-cystine residues, the search for an i n t e r c h a i n disulfide bridge was again c a r r i e d out. F r a c t i o n C~ was either oxidized or S-alkylated after reduction, then the modified sample ap p l i ed on the same column of S e p h a d e x G-100. T h e elution profile (Fig. 3) shows 2 distinct peaks, w h i c h suggests that the p e p ti d e in fraction Cz consists of 2 fragments linked by a disulfide bond. The peptides w e r e named, a c c o r d i n g to t h e i r o r d e r of elution, peptide III (Ve/Vo = 2.46) and IV (Ve/Vo = 2.86), and analyzed for t h ei r am i n o acid content and end groups (Tables II and III). No h o m o s e r i n e w a s detected in peptide III and a leucine residue was found at the C-terminal position. This finding was i n d i c a t i v e of the locaBIOCHIMIE,

1971, 53, n ° 9.

I 0.3-

o

.1.o

I

I

0.2-

W" I 2 ~

W"

I

1Vo

I

0.1

2

2Vo

E LUTIONVOLUME

FIG. 4. Gel filtration of fractions D and E on a (2.5 × 100 cm) column of Sephadex G-75 (fine). The elution conditions are the same as in Fig. 2 with the exception of the flow rate (21) ml/h) and the void volume (I47 ml). -

-

P ep t i d e V was c h a r a c t e r i z e d as usual by am i n o acid analysis and end group d e t e r m i n a t i o n (Tables 1I and III). F u r t h e r m o r e , since 2 halfcystine residues w e r e present, the search for a possible i n t e r p e p t i d i c disulfide bridge was c a r r i e d out by analyzing oxidized fractions. No splitting of p ep t i d e V w as observed. In a d d i t i o n only one serine residue was found at the N-terminal post-

964

P . C o z z o n e , L . P a s d r o , B. B e a u p o i I a n d G. M a r c h i s - M o u r e n .

tion. Fraction p e p t i d e VI.

D e w a s f o u n d to c o n t a i n

mostly

F r a c t i o n E. - - T h i s f r a c t i o n w a s also f r a c t i o n a t e d o n S e p h a d e x G-75 to give 3 m a j o r p e a k s El, E 2 a n d E a (Fig. 4). F r a c t i o n E 1 c o n t a i n s o n l y o n e c o m p o u n d i d e n t i c a l to t h e o n e f o u n d i n f r a c t i o n D 1. T h e m a j o r c o m p o n e n t in f r a c t i o n E 2 w a s f u r t h e r p u r i f i e d o n S e p h a d e x G-100 a n d y i e l d e d a s i n g l e s y m m e t r i c a l p e a k ( V e / V o = 2.46) c o n t a i n i n g p u r e p e p t i d e VI. T h i s p e p t i d e w a s r o u t i n e l y a n a l y z e d . I n p a r t i c u l a r , p r o l i n e w a s f o u n d at the N-terminal position and only one half-cystine residue was found in the chain, which obviously c o r r e s p o n d s to o n e of t h e 2 f r e e s u l f h y d r y l g r o u p s found in the intact amylase molecule. F r a c t i o n F. - - T h r e e c o m p o n e n t s w e r e f o u n d i n t h i s f r a c t i o n (Fig. 5). T h e i r s e p a r a t i o n w a s a c h i e v e d b y a s e r i e s o f 2 f i l t r a t i o n s ( T a b l e I). T h e first f i l t r a t i o n o n S e p h a d e x G-50 (fine) gave 3 p e a k s F1, F 2 a n d Fz, all f o u n d still i m p u r e . E a c h fraction was refiltered after pooling with approp r i a t e n e i g h b o r i n g f r a c t i o n s ( T a b l e I). F~ w a s p o o l e d w i t h E:~ a n d c h r o m a t o g r a p h e d t h r o u g h a S e p h a d e x G-75 c o l u m n , 2 p e a k s w e r e e l u t e d a n d c h a r a c t e r i z e d r e s p e c t i v e l y as p e p t i d e VI a n d VII. F r a c t i o n F., w a s p u r i f i e d o n S e p b a d e x G-25

(Fig. 6). T h e m a i n p e a k o b t a i n e d ( V e / V o = 1.38) w a s a n a l y z e d a n d f o u n d to b e p u r e ( p e p t i d e VII). D u e to t h e a b s e n c e of t r y p t o p h a n , t h e 280 n m a b s o r p t i o n of t h i s p e p t i d e is p a r t i c u l a r l y l o w a s c o m p a r e d to its n i n h y d r i n profile. P e p t i d e V I I is also c h a r a c t e r i z e d b y its l a c k of h a l f - c y s t i n e r e s i d u e . T h e l a s t p e a k , Fa, h a d a c o m p o s i t i o n s i m i l a r to f r a c t i o n G 2 a n d w a s t h e n p o o l e d w i t h t h i s f r a c t i o n in t h e c o u r s e of p u r i f i c a t i o n of p e p t i d e VIII. F r a c t i o n G. - - T h e f i l t r a t i o n p a t t e r n of t h i s f r a c t i o n o n S e p h a d e x G-50 s h o w s a t l e a s t 3 c o m p o n e n t s (Fig. 5). F r a c t i o n G 1 ( V e / V o ----- 1.40) h a s a w e a k 280 n m a b s o r p t i o n a n d w a s f o u n d to contain mostly peptide VII: this fraction was

.1.0

I~

.0.5

I

I

I

I

i

0.3 ~

0.2

E

0.1

tO E "E

0

0o

£N

I G21 I

1.0

Fraction 6 2

I G22

1

=: c v >-

I,:~

j%

I.I

F r a c t i o n F2

I" t

~.

I:"1

I

.1.0

I

Fraction F 0.3"

/

0.2-

~O.

O.1

G,

G2

I

i

I

I

i

o

Fraction H 2

I

G3

I--

o

,

.tO

_o

H2~

1.0

0.2

-0.5

I

Fraction G

.1 0.3

.05

///--~\\

0.2 - E

Lu 10

Fraction H

/ ~

H2

I I

II

H~

I

~ /

"

'

x

.0,5

~

0.3

t

I

2vo ELUTION

01

Hj

I

1v0

?

0.2

VOLUME

FI6. 6. - - R e f r a c t i o n a t i o n of fractions F~, G~ a n d H2 on a (1.5 × 90 em) column of Sephadex G-25 (fine). The column was developed by descending, elution. The elution conditions are the same as in r i g . 2, except for the flow rate (8 ml), the void volume (60 ml) and the volume of the fractions collected (2 ml). ( ) 280 n m absorbance ; ( ......... ) 570 n m n i n h y d r i n reaction absorbance.

1 I

1Vo

I

1.5Vo

i

\l

2V o 25\ ELUTION VOLUME

F]c,. 5. - - Gel filtrations of fractions D, G and H on a (2.5 × 10r0 cm) column of Sephadex G-50 (fine). The elution conditions are the same as in Fig. 2 w i t h the exception of the flow rate (25 m l / h ) and the void volume (117 mt). ( ) 280 nm absorbance ; ( ........ ) 570 nm n i n h y d r i n reaction absorbance. BIOCHIMIE, 1971, 53, n ° 9.

t h e n p o o l e d w i t h f r a c t i o n F 2. F r a c t i o n G 2 w a s f u r t h e r p u r i f i e d b y f i l t r a t i o n o n S e p h a d e x G-25 (Fig. 6). A n a l y s i s of t h e m a i n p e a k ( V e / V o : 1.70) i n d i c a t e s a p u r e c o m p o n e n t ( p e p t i d e VIII). T h i s p e p t i d e is e s p e c i a l l y c h a r a c t e r i z e d b y its e n d groups: an N-terminal blocked group and a

Cyanogen

bromide

peptides

C-terminal homoserine. Identification of an acetyl group as b l o c k i n g the N-terminal peptide position was u n a m b i g u o u s l y demonstrated by Nuclear Magnetic Resonance [24]. The third peak (fraction Ga) behaves chromatographically like the n e i g h b o r i n g fraction H 2 (Fig. 5). Moreover, its end groups and amino acid composition are similar to this neighboring fraction ; therefore both fractions G:; and H e were pooled. F r a c t i o n H. - - Gel filtration analysis of fraction H reveals at least 3 components Ha, H e and H z (Fig. 5). End group analysis of H a gave no detectable N-terminal residue by dansylation and the a m i n o acid composition was identical to that of peptide VIII. The major peak H 2 was purified further on Sephadex G-25 (Fig. 6). Pure peptide IX w a s thus obtained. Amino acid analysis of this peptide indicates no half-cystine residue ; however, the n u m b e r of aromatic residues in peptides IX is very high (9 residues out of 26). This explains why, although the molecular weight of peptides VIII and IX are almost the same, their c h r o m a t o g r a p h i c beheviour on Sephadex is quite different, due to the high affinity of Sephadex for aromatic structures. F r a c t i o n M e . - - In some cases a n i n h y d r i n positive fraction called M2 was observed at the very end of the elution pattern (Fig. 2). It never contained more than 0.5 p. cent in weight of the total material and was ascertained as consisting of some degradation products.

The c h r o m a t o g r a p h i c b e h a v i o u r of homologous CNBr-peptides from both molecular forms of amylase was found quite similar. The molecular weight, the amino acid composition and the end groups were also d e t e r m i n e d for both series of peptides : identical results were oblained. DISCUSSION AND CONCLUSION. Structural problems,

W h e n setting our cleavage c o n d i t i o n s two main r e q u i r e m e n t s had to be met. First, the cleavage had to be complete, second, the splitting had to be specific for methionine. Completion of the cleavage is shown in Fig. 1. Moreover, analysis of the aggregate contained in F r a c t i o n M~ did not reveal the presence of any uncleaved material. It shouht be noticed, however, that 7 out of 8 m e t h i o n i n e residues appear to react in a few hours w h i l e the conversion of the last residue requires m u c h more time. One explanation is that this m e t h i o n i n e residue is linked with the serine residue found in the N-terminal position of peptide V. Actually, the slow cleavage of Met-Ser BIOCHIMIE, 1971, 5 3 ,

n ° 9.

o{ p a n c r e a t i c

amylase.

965

as well as Met-Thr bond would be a result of the p a r t i c i p a t i o n of the h y d r o x y l group in an intermediate reaction [25]. CNBr is also k n o w n to react with t r y p l o p h a n [26] but such a reaction does not occur u n d e r our conditions since the sum of t r y p t o p h a n residues found in the CNBrpeptides is equal to the n u m b e r of residues determ i n e d in the entire amylase molecule. A possible side reaction of 70 p. cent trifluoroacetic acid with amylase has also been investigated. The control e x p e r i m e n t gave no i n d i c a t i o n of any such chemical alteration. The overall procedure (Table I) used for peptide purification may deserve some c o m m e n t : amylase has been treated with CNBr directly w i t h o u t any p r i o r chemical modification. Especially, disulfide bridges were not split before CNBr cleavage. This was not necessary since only 1 out of the 4 disulfide bridges was found to link 2 p o l y p e p t i d i c chains in the CNBr cleavage mixture. F r o m the a m i n o acid composition data of the peptides (Table III), the location of the other bridges is easily deduced as well as the position of the freeSH groups. Two disulfide bridges are contained in peptide I, 1 brige is present in peptide V and the 4th one has been shown to link peptide III to peptide IV. The 2 free-SH groups already detected i n the entire molecule [3] are u n a m b i guously located in peptides I1 and VI. Actually, a d i s r u p t i o n of the S-S b o n d s would have even r e n d e r e d the purification problem more difficult to solve : 2 additional small peptides (III and IV) would have been p r e s e n t in the initial digestion mixture, w i t h a molecular weight in the same range as for peptide VI, VIII and IX. Disulfidelinked peptides III and IV were thus purified as such, before cleavage and final separation one from the other. Moreover, the initial splitting and blocking of the disulfide bridges in the entire amylase molecule might have i m p a i r e d a subsequent complete cleavage at the m e t h i o n i n e level since this residue has been found to be sensitive either to oxidation [10] or to alkylating agents [27-291. Ordering of the peptides to reconstitute the amylase molecule is, of course, not possible at this stage except for the N- and C-terminal peptides. These 2 t e r m i n a l peptides can be distinguished by their end groups. The C-terminal peptide (peptide III)Moes not contain homoserine and its C-terminal residue (l.eucine) is the same as for amylase. The N-terminal peptide (VIII) in our case is also easily detected by its N-terminal blocked end already found in the entire molecule. It should be now pointed out that characterization of the N-acetyl blocking group by N.M.R. w h i c h

P. Cozzone, L. Pasdro, B. Beaupoii and G. Marchis-Mouren.

966

was not possible on the entire amylase molecule because of its large size, w a s c a r r i e d out on p ep t i d e VIII [24].

sh o w n [3] to be closely related since t h ei r enzymatic activity, m o l ecu l ar weight and end groups w e r e found identical.

An o t h er conclusion to be d r a w n from these results, c o r r o b o r a t e s the tinding of p o r c i n e pancr eat i c amylase as being a m o n o m e r i c e n z y m e [3]. Since only 1 N-terminal p e p ti d e and 1 C-terminal peptide w e r e found and since the sum of the amino acid in these 9 peptides accounts for the n u m b e r of amino acid in the w h o le molecule, the amylase molecule should only consist of a single p o l y p e p t i d e chain. Should 2 different chains be present, 10 peptides would have been liberated instead of 9. Supposing 2 identical chains exist, only 5 peptides would have been obtained ( ' ) .

The am i n o acid compositions are si m i l ar although m i n o r differences in largely r ep r esen t ed residues might not have been detected w h e n comp a r i n g the amino acid co m p o si t i o n of the entire amylase molecules, because of limited sensitivity in the analysis. R e m a r k a b l y a significant 4-6 aspartic acid a n d / o r asparagine residues difference can be r o u t i n el y observed. In addition, based on c h r o m a t o g r a p h i c and e l e c t r o p h o r e t i c behavior, amylase II (pI = 5.25), appears to be more a c i d i c than amylase I (pI = 5.95) [30]. A more p r eci se structural c h a r a c t e r i z a t i o n of these two forms was expected from the compari-

TABLE IV.

Schematic representation of porcine pancreatic (x-amylase. The order of the 7 peptides in the parentheses is, of course, arbitrary. Only the N and C-terminal peptides are positioned.

@

@ Acetyl

@ HSer I

HSer[ Val, I

I

I

S--S

® Pro

SH

@ HSer

Ser

,

Leu HSer I I

I

I

S~S

Val

'

HSer I

/

The 2 amylase forms problem.

Val '

s

Leut I

Multiple forms of enzymes may either be due to differences in the p r i m a r y structure, w h i c h w o u l d suggest 2 separate genes c o d i n g for these enzymes or to modification o c c u r i n g i n d e p e n d e n t l y of protein synthesis such as sugar a d d i t i o n or deamidation of glutamine and asparagine residues ; both situations may also o c c u r as in the case of bovine d e o x y r i b o n u c l e a s e s A and C [331. The v a r i a b i l i t y of the sugar moiety w h i c h has been s h o wn to be present in amylases I and II [31J could a c c o u n t for the existence of the 2 forms. On the other hand the results p u b li s h e d in the present p a p e r do not exclude any differences in the p r i m a r y structure. Amylases I and II have been

®

HSer

~

1$

BIOCHIMIE, 1971, 53, n ° 9.

ValI

S--S

® HSer

~

I

@

"!

HSer/ I

SH

®

'

J

Lys SerAlaGlySerlleValTyr PheLEU

son of the CNBr-peptides obtained by separate cleavage of amylase I or II. Surprisingly, no significant difference in the m o l e c u l a r weight, amino acid co m p o si t i o n and am i n o and c a r b o x y l terminal residues w as found w h e n c o m p a r i n g the homologous peptides. In o t h er w o r d s even the difference of 5 aspartic acid residues seen between the 2 complete molecules does not a p p e a r at the peptide level. To u n d e r s t a n d this a p p a r e n t d i s c r e p a n c y 2 hypotheses will now be discussed. Co n si d er i n g the position in the amylase I ch ai n of these 5 (') Our results are in contradistinction vcith the recent work by ROBYTet al [35] assumin~ the existence of 2 identical chains.

Cyanogen

bromide

peptides

of pancreatic

amylase.

967

e x t r a r e s i d u e s , 2 e x t r e m e c a s e s m a y be c o n s i d e r e d : t h e a s p a r t i c a c i d c o r r e s p o n d i n g to m u t a t e d o r d e l e t e d p o s i t i o n m a y be e i t h e r s c a t t e r e d in t h e v a r i o u s C N B r - p e p t i d e s o r at t h e o p p o s i t e , g r o u p e d as a c l u s t e r in o n l y o n e r e g i o n of t h e a m y l a s e I m o l e c u l e . I n t h e tirst c a s e d u e to t h e u n c e r t a i n t y in t h e a m i n o a c i d c o m p o s i t i o n of t h e l a r g e p e p t i d e s the d i f f e r e n c e in t h e n u m b e r of aspartic acid residues may not have been detected at t h e p e p t i d e level. In t h e c a s e of an a s p a r t i c a c i d c l u s t e r t h e d i f f e r e n c e s h o u l d of c o u r s e be a p p ' w e n t . A l t h o u g h t h i s is n o t t h e case, t h e c l u s t e r h y p o t h e s i s is still valid b y s u p p o s i n g t h a t t h e 5 a s p a r t i c a c i d r e s i d u e s h a v e b e e n lost in t h e c o u r s e of t h e p u r i f i c a t i o n of t h e C N B r - p e p t i d e s . A c t u a l l y a s p a r t i c a c i d b o n d s a r e k n o w n to he l a b i l e w h e n i n c u b a t e d in a c i d m e d i u m at h i g h t e m p e r a t u r e [32].

tiques ; leur composition en amino acide ne diff~re que sur la teneur en acide aspartique e t / o u asparagine. Dans le present travail, une comparaison plus precise des 2 amytases est obtenue p a r compa~aison des peptides r4suttant du clivage de l'amylase par le b r o m u r e de cyanog~ne. Comme le montre la cin4tique de elivage, ]es conditions utilis~es p e r m e t t e n t une coupure totale et sp~cifique au nivean des m~thionines. Les 9 peptides attendus sont purifi~s par tamisage mol~eulaire et earact4ris4s par leur poids mol~culaire, leurs extr~mit4s terminales et leur composition en amino aeide. La position des 4 p o r t s dis~lfure et celle des 2 thiols libres s o r t ~galement pr~eis~es. Les peptides N et G-terminaux sont identifi4s. La eomparaison structurale entre peptides homologues obtenus /t partir des amylases I e t I I n e r~v61e aueune difference. En particulier, la teneur en acide aspartique e t / o u asparagine des peptides homologues est identique, Ces r6sultats sont diseut4s.

F r o m t h e e n d g r o u p d e t e r m i n a t i o n of t h e p e p t i d e s it a p p e a r s t h a t a p o s s i b l e l o c a t i o n f o r an a s p a r t i c r e s i d u e c l u s t e r w o u l d be at t h e N - t e r m i nal e n d of an i n t e r n a l C N B r - p e p t i d e . At t h e moment the only indication favouring this hypot h e s i s is t h e r e p r o d u c i b l e f i n d i n g o f s m a l l a m o u n t s of d a n s y l - a s p a r t i c a c i d b e s i d e d a n s y l s e r i n e w h e n a n a l y z i n g p e p t i d e V. H o w e v e r , m i n o r a m o u n t s of d a n s y l a s p a r t i c a c i d a r e f o u n d as w e l l in p e p t i d e V d e r i v e d f r o m a m y l a s e I as in t h e o n e o b t a i n e d f r o m a m y l a s e II. T h i s a s p a r t i c a c i d c l u s t e r m i g h t also be l o c a t e d at t h e N - t e r m i n a l p o s i t i o n of p e p t i d e VI. A c t u a l l y t h e A s x - P r o b o n d is k n o w n to be labile in c o n c e n t r a t e d o r g a n i c a c i d I34!. T h e a s p a r t i c a c i d c l u s t e r c o u l d t h u s b e detached w h e n the f r a c t i o n s are c o n c e n t r a t e d by e v a p o r a t i o n at a b o u t 40 °, b e f o r e r e f i l t r a t i n g t h e peptides.

Die aus dem Schweinspankreas gereinigten Amylasen I und II haben dieselbe enzymatisehe Aktivit~it ; ihr Molekulargewicht und ihre Endextremit~iten sind identisch ; ihre Aminos~iurezusammensetzung unterseheidet sich nur dureh den Gehalt an Asparaginsiiure u u d / o d e r an Asparagin. In der vorliegenden Arbeit wird ein genauerer Vergleich der durch Spalten der Amylase mittels Cyanogenbromid entstehenden Peptide erhalten. Wie es die Kinetik zeigt, erl.anben die benutzten Bedingungen ein vollst~indiges und spezifisehes Spalten auf der Stufe der Methionine. Die 9 erwarteten Peptide werden durch Molekularsieben gereinigt und dutch ihre Molekulargewichte, ihre Endextremit~iten und ihre Aminos~iurezusammensetzung gekennzeichnet. Die Stelle der 4 Disulfidb,rficken und diejenige der 2 freien Thiole werden ebcnso genauer angegeben. Die N- und C-Endpeptide vcerden identiflziert. Der Strukturvergleich zwischen homologen Peptiden, welche aus den Amylasen I und II erhalten werden, zeigt keinen Unterschied. Insbesondere ist der Gehalt an Asparagins~iure u n d / o d e r Asparagin der homologen Peptide identiseh. Diese Ergebnisse werden besprochen.

W h a t e v e r t h e fate of t h e a s p a r t i c a c i d r e s i d u e s , further c h a r a c t e r i z a t i o n of the CNBr-peptides has to be c a r r i e d out in o r d e r to e l u c i d a t e t h e s t r u c tural differences between the 2 amylase forms.

Acknowledpmen Is. We wish thank Prof. P. DESNUELLE for continuous interest throughout the course of this work, an,d Miss Ch. TEISSIER for her skillful technical assistance. The work described was supported by a grant of the C.N.R.S. (E.R.A. n ° 173). For one of us (P. COZZONE) most of this work was done in partial fulfillment of the requirements for the degree of Doeteur-~s-Sciences, Universit~ de Provence (Thesis C.N.R.S.-A.O. 4229).

RgSUM~. Les amylases I e t It purifi~es h partir du pancr4as de pore ont mgme activitg enzymatique ; leur poids mol6eulaire et leurs extrgmitgs terminales sont iden-

BIOCHIMIE, 1971, 53, n ° 9.

ZUSAMMENFASSUNG,

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