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The tryptic peptide with the rennin-sensitive linkage of cow's κ-casein
591
B I O C H I M I C A ET B I O P H Y S I C A A C T A
PRELIMINARY NOTES BBA
31049
The tryptic peptide with the rennin-sensitive linkage of cow's K-casein* Many detailed chemical structure studies have been devoted to the blood clotting process, but only a few data are available in the field of the nfilk clotting process. The primary enzymic phase of the action of rennin (EC 3.4.4.3) on casein I in which the protective colloid is destroyed has been shown to be confined to the K-casein fraction 2. During this phase, rennin liberates from cow's K-casein at pH 6.8 a glyeopeptide which we called K-caseino-glycopeptide, and even in the absence of I;a 2+, the remaining part of K-casein, the so called para-K-casein, precipitates. The C-terminal sequences of K-caseino-glycopeptide, as well as of K-casein, were reported by JOLLIES, ALAIS AND JOLL~S3 to be the same; therefore it may be assumed that K-caseinoglycopeptide is situated at the C-terminal end of K-casein. In agreement with these results are the facts that after rennin was allowed to act on K-casein, no new N-terminal amino acids could be detected in the para-K-casein produced a, and a new N-terminal residue, methionine, was found by our group in the K-easeino-glycopeptide4. We described later the N-terminal octadecapeptide of K-caseino-glycopeptide: Met-AlaIle-Pro-Pro--Lys-Lys-Asn-(Asx, Glx)-Lys-Thr-Glu-(Pro, lie) (Asn, Thr, lie) (ref. 5). A new C-terminal amino acid, phenylalanine, which came from the pars-K-casein, was found following rennin action 3. The question arises whether it can be assumed that a Phe-Met linkage has been cleaved and therefore it seemed important to localize in K-casein the sequence containing this linkage. An attempt was thus made to isolate a large tryptic peptide from K-casein possessing r e s t - P h e . . . Met Ala-lie-Pro P r o - L y s as its C-terminal sequence. It is worth mentioning that K-casein seems to have a homogeneous peptide skeleton in the para-K-casein moiety (J. JOLL~S, unpublished results); a few changes attributable mainly to genetic variants are situated in the K-caseino-glycopeptide part. These genetic variants and the variations in the sugar composition are ultimately responsible for the heterogeneity observed upon electrot)horesis 6. The chromatography on Dowex I-X2 of the tryptie digest of reduced aminoethylated cow's K-casein prepared according to Mc KEXZIE AND WAKE 7, gave rise to 22 peaks. The analysis of Peak I before purification indicated the presence of Phe, Met, Pro and Lys among many other amino acids; this peak was thus capable of containing a sequence with a Phe-Met bond; its purification was performed also because the second Met residue s of K-casein was found in a tryptie peptide containing Arg (J. JOLL~S, unpublished results) which is not present in the C-terminal peptide of K-casein. Two peaks of the tryptic digest were devoid of basic amino acids: Peak 21, the amino acid composition of which corresponded to the C-terminal moiety of Kcaseino-glycopeptide and represented, in fact, the C-terminal sequence of K-casein, and Peak 9. Because preliminary experiments indicated that the rennin-sensitive linkage of K-casein was also sensitive to other enzymes 6, the study of this peptide was also undertaken because it could be situated at the C-terminal side of para-K-casein. * i9th communication on caseins: iSth communication, A. M. FIAT, C. ALMSAND P. JOLL~S, Chimia, >tarau, 22 (1968) 137.
Biochim. Bwphys. Acta, 168 (I9(~8) 591 593
PI
592 T , \ I3LE 1 S'ITI~.U(TldRI';
OF
Ibl
PFPTIDI~;S
AND
9 [)
C o m p o s i t i o n : Sert, l'ro.2, ].cut, l>het , lIis.a. .'llethod
Isolated st~bsla~zces
F~dman Partial hydrolysis
His (His, Ix:u) S e t l)he
Carboxypeptidase : 2 rain
~er
] qlO
(2o%) (0s%) I o rain Structure
l.eu Ser l ' h e t f i s l ' r o Pro His l.eu Ser P h e
Peak z (Dowex I-X~). The rechromatography of Peak I on Dowex 5o-X2 (x2 cm × o. 9 cm) gave rise to 3 main peptides (Peptides Ia-Ic). Peplide zb. Amino acid composition: Ser l, Pr%, Ala 1, Met> Ilel, Leu l, Phe 1, His,~, Lys x. After chymotryptic digestion, two peptides (IbI and IbII) were obtained and isolated by preparative paper chromatography. Peplide zbI: it contained no basic amino acid and nmst therefore be the N-terminal sequence of tryptie Peptide Ib. The establishment of its structure is summarized in Table I. Peptide zbII: it contained a lysine residue and must therefore be situated at the C-terminal side of tryptic Peptide ib. Amino acid composition: Pro> Alal, Metl, I1%, Lys 1. It corresponded to the Nterminal sequence of K-caseino-glycopeptide which had already been studied in detail by DELFOUR, M.Ms AND JOLLL;S'LThe formula of Peptide I1) is indicated in Scheme I. Pefllide so. This peptide had the same amino acid composition as Peptide Ib with one exception : it contained two lysine residues. Our previous studies have established 5 that the N-terminal sequence of K-caseino-glycopeptide was Met Ala Ile Pro--Pro Lys Lys and that the tryptic digestion gave rise to the peptides with one and two lysine residues. It was therefore possible to conclude that Peptides Ib and ic had the same structures, with one additional lysine residue in Peptide ic (see SchemeI). Peak 9 (Dowex I-Xe). The rechromatography of Peak 9 on Dowex 5o-Xe (3o c m x 0.9 cm) gave rise to two main peptides (Peptides 9 a and 9b). Peptide 9 b. This peptide contained no basic amino acid. Its amino acid composition aim structure
T II . . . . . . . . . . . .
Lys
His
TPru
Pro
Ilis
gt,tt St!r
Phe
-
-1,,n-Mt,t AIa
Ile
prn
--+T Pro
LysIl,v~
T .....
V a l . O11
I
l'(,I/[i ill --llnra-~c-caspii/ ,If
~ G-C~tNOill
~, - C a S t ~ i i l o - K l y c < q o c p l i d t ,
-~ ~1~
S c h e m e 1. T h e s e q u e n c e c o n t a i n i n g t h e r e n n i n - s e n s i t i v e l i n k a g e in c o w ' s g casein. T, t r y p t i c s p l i t . Bi<>chim. 1~7ophys. ,4cia, lOS (1908) 5 9 t 593
593
P R E L I M I N A R Y NOTE
were identical to those of Peptide i b I (Table I). All these data indicate that Peptide 9 b corresponds to the N-terminal moiety of Peptide I b which is the overlapping peptide of para-K-casein and K-caseino-glycopeptide (Scheme I); Peptide 9 b is also the Cterminal sequence of para-K-casein and contains the shorter sequence keu-Ser Phe already described by DE KONINC;~. Previously it was suggested that rennin ndght be splitting an ester linkage rather than a peptide linkage. The present results are not in favour of such an interpretation. They exclude also the presence of a peptide between the C-terminal Phe residue of para-K-casein and the N-terminal Met residue of K-caseino-glycopeptide which might have been lost when the rennin digest was dialyzed in view of purifying the K-caseino-glycopeptide. The linkage split in K-casein by rennin is a very particuliar one, as it is sensitive to m a n y enzymes 6,9 such as trypsin, chymotrypsin, pepsin, kallicrein and also to nonenzymic treatments as LiBH 4 and heaO °. Tryptic Peptide I b (Scheme I) is however not digested by rennin in the conditions applied to native K-casein, but it is always sensitive to chymotrypsin. We were still unable to obtain the complete degradation of Peptide Ib by E r d m a n ' s method having in view the determination of the Peptidic Phe-Met linkage by another procedure. It is worth pointing out that this peptide has very high contents of proline and histidine. The presence of two histidine residues seems in accordance with data of HILL AXl~ LAINGn who reported tim loss of the ability of K-casein to be split and clotted by rennin after photooxidation in the presence of methylene blue. These authors concluded that both these effects were caused by alteration of histidine residues and that these latter m a y be part of the site of rennin action on K-casein.
Laboratory of Biochemistrx~ Faculty of Sciences, 96 Bd. Raspail Paris* and Dairy Department**, Nancy (France) I 2 3 4 5 6 7 8 9 to Ii
JACQUELINEJoLI.kS* C H A R L E S AI.AIS** PIERRE
JOLLY,S*
C. ALAIS, G. MOCQUOT, H. NITSCHMANN AND i'. ZAHLER, Helv. Chim. Acta, 36 (1953) 1955. D. F. WAUGH AND P. H. VON HIPPEL, J. Am. Chem. Soc., 78 (t956) 4576 . 1'. JOLL~S, C. ALAIS AND J. JOLL~S, Arch. Biochem. Biophys., 98 (1962) 56 . A. DELFOUR, J. JOLL/~S, C. ALAIS AND 1'. JOLL~S, Bioehem. Biophys. Res. Commun., i 9 (196.5) 452. A. DELFOUR, C. ALMS AND l'. JOLLY;S, Chimia, .4arau, 20 (196(,) 148. P. JOLL~S, Angew. Chem. lntern. Ed. Engl., 5 (1966) 558. H. A. M c I'~ENZlF~ AND R. G. \VAKE, Biochim. Biophys. ~4cta, 47 (I96~) 24o. H. A. M c NE~ZlE, Advan. Protein Chem., 22 (1967) 122. 1~- J. DE KONING, Thesis, A m s t e r d a m , 1967 . C, ALAIS, N. NIGER AND P. JOLLgS, J. Dairl, Sci., 5 ° (1967) i778. R. D. HILL AND R. R. LAING, .f. Dairy Res., 3 ° (1963) 375.
Received July 29th, I968 Biochim. Biophys. ,4clot, 168 (1968) 5 9 1 - 5 9 3
B I O C H I M I C A ET B I O P H Y S I C A A C T A
PRELIMINARY NOTES BBA
31049
The tryptic peptide with the rennin-sensitive linkage of cow's K-casein* Many detailed chemical structure studies have been devoted to the blood clotting process, but only a few data are available in the field of the nfilk clotting process. The primary enzymic phase of the action of rennin (EC 3.4.4.3) on casein I in which the protective colloid is destroyed has been shown to be confined to the K-casein fraction 2. During this phase, rennin liberates from cow's K-casein at pH 6.8 a glyeopeptide which we called K-caseino-glycopeptide, and even in the absence of I;a 2+, the remaining part of K-casein, the so called para-K-casein, precipitates. The C-terminal sequences of K-caseino-glycopeptide, as well as of K-casein, were reported by JOLLIES, ALAIS AND JOLL~S3 to be the same; therefore it may be assumed that K-caseinoglycopeptide is situated at the C-terminal end of K-casein. In agreement with these results are the facts that after rennin was allowed to act on K-casein, no new N-terminal amino acids could be detected in the para-K-casein produced a, and a new N-terminal residue, methionine, was found by our group in the K-easeino-glycopeptide4. We described later the N-terminal octadecapeptide of K-caseino-glycopeptide: Met-AlaIle-Pro-Pro--Lys-Lys-Asn-(Asx, Glx)-Lys-Thr-Glu-(Pro, lie) (Asn, Thr, lie) (ref. 5). A new C-terminal amino acid, phenylalanine, which came from the pars-K-casein, was found following rennin action 3. The question arises whether it can be assumed that a Phe-Met linkage has been cleaved and therefore it seemed important to localize in K-casein the sequence containing this linkage. An attempt was thus made to isolate a large tryptic peptide from K-casein possessing r e s t - P h e . . . Met Ala-lie-Pro P r o - L y s as its C-terminal sequence. It is worth mentioning that K-casein seems to have a homogeneous peptide skeleton in the para-K-casein moiety (J. JOLL~S, unpublished results); a few changes attributable mainly to genetic variants are situated in the K-caseino-glycopeptide part. These genetic variants and the variations in the sugar composition are ultimately responsible for the heterogeneity observed upon electrot)horesis 6. The chromatography on Dowex I-X2 of the tryptie digest of reduced aminoethylated cow's K-casein prepared according to Mc KEXZIE AND WAKE 7, gave rise to 22 peaks. The analysis of Peak I before purification indicated the presence of Phe, Met, Pro and Lys among many other amino acids; this peak was thus capable of containing a sequence with a Phe-Met bond; its purification was performed also because the second Met residue s of K-casein was found in a tryptie peptide containing Arg (J. JOLL~S, unpublished results) which is not present in the C-terminal peptide of K-casein. Two peaks of the tryptic digest were devoid of basic amino acids: Peak 21, the amino acid composition of which corresponded to the C-terminal moiety of Kcaseino-glycopeptide and represented, in fact, the C-terminal sequence of K-casein, and Peak 9. Because preliminary experiments indicated that the rennin-sensitive linkage of K-casein was also sensitive to other enzymes 6, the study of this peptide was also undertaken because it could be situated at the C-terminal side of para-K-casein. * i9th communication on caseins: iSth communication, A. M. FIAT, C. ALMSAND P. JOLL~S, Chimia, >tarau, 22 (1968) 137.
Biochim. Bwphys. Acta, 168 (I9(~8) 591 593
PI
592 T , \ I3LE 1 S'ITI~.U(TldRI';
OF
Ibl
PFPTIDI~;S
AND
9 [)
C o m p o s i t i o n : Sert, l'ro.2, ].cut, l>het , lIis.a. .'llethod
Isolated st~bsla~zces
F~dman Partial hydrolysis
His (His, Ix:u) S e t l)he
Carboxypeptidase : 2 rain
~er
] qlO
(2o%) (0s%) I o rain Structure
l.eu Ser l ' h e t f i s l ' r o Pro His l.eu Ser P h e
Peak z (Dowex I-X~). The rechromatography of Peak I on Dowex 5o-X2 (x2 cm × o. 9 cm) gave rise to 3 main peptides (Peptides Ia-Ic). Peplide zb. Amino acid composition: Ser l, Pr%, Ala 1, Met> Ilel, Leu l, Phe 1, His,~, Lys x. After chymotryptic digestion, two peptides (IbI and IbII) were obtained and isolated by preparative paper chromatography. Peplide zbI: it contained no basic amino acid and nmst therefore be the N-terminal sequence of tryptie Peptide Ib. The establishment of its structure is summarized in Table I. Peptide zbII: it contained a lysine residue and must therefore be situated at the C-terminal side of tryptic Peptide ib. Amino acid composition: Pro> Alal, Metl, I1%, Lys 1. It corresponded to the Nterminal sequence of K-caseino-glycopeptide which had already been studied in detail by DELFOUR, M.Ms AND JOLLL;S'LThe formula of Peptide I1) is indicated in Scheme I. Pefllide so. This peptide had the same amino acid composition as Peptide Ib with one exception : it contained two lysine residues. Our previous studies have established 5 that the N-terminal sequence of K-caseino-glycopeptide was Met Ala Ile Pro--Pro Lys Lys and that the tryptic digestion gave rise to the peptides with one and two lysine residues. It was therefore possible to conclude that Peptides Ib and ic had the same structures, with one additional lysine residue in Peptide ic (see SchemeI). Peak 9 (Dowex I-Xe). The rechromatography of Peak 9 on Dowex 5o-Xe (3o c m x 0.9 cm) gave rise to two main peptides (Peptides 9 a and 9b). Peptide 9 b. This peptide contained no basic amino acid. Its amino acid composition aim structure
T II . . . . . . . . . . . .
Lys
His
TPru
Pro
Ilis
gt,tt St!r
Phe
-
-1,,n-Mt,t AIa
Ile
prn
--+T Pro
LysIl,v~
T .....
V a l . O11
I
l'(,I/[i ill --llnra-~c-caspii/ ,If
~ G-C~tNOill
~, - C a S t ~ i i l o - K l y c < q o c p l i d t ,
-~ ~1~
S c h e m e 1. T h e s e q u e n c e c o n t a i n i n g t h e r e n n i n - s e n s i t i v e l i n k a g e in c o w ' s g casein. T, t r y p t i c s p l i t . Bi<>chim. 1~7ophys. ,4cia, lOS (1908) 5 9 t 593
593
P R E L I M I N A R Y NOTE
were identical to those of Peptide i b I (Table I). All these data indicate that Peptide 9 b corresponds to the N-terminal moiety of Peptide I b which is the overlapping peptide of para-K-casein and K-caseino-glycopeptide (Scheme I); Peptide 9 b is also the Cterminal sequence of para-K-casein and contains the shorter sequence keu-Ser Phe already described by DE KONINC;~. Previously it was suggested that rennin ndght be splitting an ester linkage rather than a peptide linkage. The present results are not in favour of such an interpretation. They exclude also the presence of a peptide between the C-terminal Phe residue of para-K-casein and the N-terminal Met residue of K-caseino-glycopeptide which might have been lost when the rennin digest was dialyzed in view of purifying the K-caseino-glycopeptide. The linkage split in K-casein by rennin is a very particuliar one, as it is sensitive to m a n y enzymes 6,9 such as trypsin, chymotrypsin, pepsin, kallicrein and also to nonenzymic treatments as LiBH 4 and heaO °. Tryptic Peptide I b (Scheme I) is however not digested by rennin in the conditions applied to native K-casein, but it is always sensitive to chymotrypsin. We were still unable to obtain the complete degradation of Peptide Ib by E r d m a n ' s method having in view the determination of the Peptidic Phe-Met linkage by another procedure. It is worth pointing out that this peptide has very high contents of proline and histidine. The presence of two histidine residues seems in accordance with data of HILL AXl~ LAINGn who reported tim loss of the ability of K-casein to be split and clotted by rennin after photooxidation in the presence of methylene blue. These authors concluded that both these effects were caused by alteration of histidine residues and that these latter m a y be part of the site of rennin action on K-casein.
Laboratory of Biochemistrx~ Faculty of Sciences, 96 Bd. Raspail Paris* and Dairy Department**, Nancy (France) I 2 3 4 5 6 7 8 9 to Ii
JACQUELINEJoLI.kS* C H A R L E S AI.AIS** PIERRE
JOLLY,S*
C. ALAIS, G. MOCQUOT, H. NITSCHMANN AND i'. ZAHLER, Helv. Chim. Acta, 36 (1953) 1955. D. F. WAUGH AND P. H. VON HIPPEL, J. Am. Chem. Soc., 78 (t956) 4576 . 1'. JOLL~S, C. ALAIS AND J. JOLL~S, Arch. Biochem. Biophys., 98 (1962) 56 . A. DELFOUR, J. JOLL/~S, C. ALAIS AND 1'. JOLL~S, Bioehem. Biophys. Res. Commun., i 9 (196.5) 452. A. DELFOUR, C. ALMS AND l'. JOLLY;S, Chimia, .4arau, 20 (196(,) 148. P. JOLL~S, Angew. Chem. lntern. Ed. Engl., 5 (1966) 558. H. A. M c I'~ENZlF~ AND R. G. \VAKE, Biochim. Biophys. ~4cta, 47 (I96~) 24o. H. A. M c NE~ZlE, Advan. Protein Chem., 22 (1967) 122. 1~- J. DE KONING, Thesis, A m s t e r d a m , 1967 . C, ALAIS, N. NIGER AND P. JOLLgS, J. Dairl, Sci., 5 ° (1967) i778. R. D. HILL AND R. R. LAING, .f. Dairy Res., 3 ° (1963) 375.
Received July 29th, I968 Biochim. Biophys. ,4clot, 168 (1968) 5 9 1 - 5 9 3