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Identification of an anionic trypsinogen in bovine pancreas
BIOCHIMICAET BIOPHYSICAACTA
499
BBA R e p o r t BBA 31104 I d e n t i f i c a t i o n o f an a n i o n i c t r y p s i n o g e n in b o v i n e p a n c r e a s
A. PUIGSERVER and P. DESNUELLE Centre de Biochimie et de Biologic Moldculaire du ~N.R.S., 31, Chemin £ Aiguier 13, Marseille (France)
(Received March 8th, 1971)
SUMMARY A strongly anionic trypsinogen has been identified in bovine pancreas with the aid of enterokinase as the activating agent. The new zymogen has been substantially purified by two chromatographies on DEAE-cellulose.
The enzymes and zymogens of the exocrine secretion of bovine pancreas have received more attention than those of any other species. They are known to consist essentially of two chymotrypsinogens, a cationic trypsinogen, an amylase, a lipase, a deoxyribonuclease, a ribonuclease, a procarboxypeptidase B and a procarboxypeptidase A. This latter has been shown to be a trimer 1 composed of the zymogen of carboxypeptidase A, a proendopeptidase similar to porcine chymotrypsinogen C (ref. 2) and a still unidentified protein. The presence of a second trypsinogen in the bovine secretion has never been reported, although two trypsinogens have already been shown to exist in rat a , pig 4 and human s pancreas. The purification of the anionic procarboxypeptidase A of bovine pancreas was observed in this laboratory to be often associated with partial activation of the proendopeptidase monomer. This undesirable activation could not always be prevented by addition of 1 mM benzamidine and, therefore, it was not likely to be caused by traces of cationic trypsin possibly carried into the anionic protein fraction by some other components. An anionic bovine trypsinogen has now been identified with the aid of highly purified enterokinase 6 as the activating agent. According to the usual purification procedure for procarboxypeptidase A (ref. 1), an aqueous extract (1800 ml) of an acetone powder (180 g) of bovine pancreas was introduced into a 6 cm x 40 cm DEAE-ceUulose (Brown, fibrous form) column equilibrated with a 50 mM Tris-HCl buffer (pH 7.0) 1 mM in benzamidine. The cationic proteins were washed out by passage of 6.'5 1 of the buffer 0.06 M in NaC1, and a first portion of the anionic proteins was eluted using a linear gradient of NaCI from Biochim. Biophys. Acta, 236 (1971) 499-502
500
BBA REPORT
0.06 to 0.25 M. After the emergence of the peak containing procarboxypeptidase A (identified through its potential activity* towards N-acetyl-L-tyrosine ethylester), a second portion of anionic components (proteins and nucleic acids) was eluted by another gradient so ajusted as to raise the NaC1 molarity from 0.25 to 1.0 in a buffer volume of 41. The fractions of the ascending limb of the sharp peak thus obtained were found to display a strong activity towards the trypsin substrate benzoyl-L-arginine ethylester after a 1-h incubation with enterokinase (approximate enzyme-protein weight ratio, 1:100) in a 50 mM Tris-HC1 buffer (pH 7.8) 50 mM in CaCI2. These fractions were shown by disc electrophoresis (15% gel, pH 8.6) to contain several protein components. Their nucleic acid content was evaluated by spectrophotometry to be 40%. The result of a similar chromatography with a slower NaC1 gradient (0.25 to 0.50 M in 41 of buffer) is indicated in Fig. la. A number of fractions with potential activity towards benzoyl-L-arginine ethylester are seen to emerge just ahead of a broad peak for an approximate NaC1 molarity of 0.33. Disc electrophoresis showed that these fractions contained a major protein component and some impurities. The broad peak on the far right was probably composed of nucleic acids, since no detectable bands were visible in the corresponding electrophoresis diagrams. Fractions 3 1 - 8 6 in Fig. 1a were pooled and precipitated at 0 ° by 80% saturated (NH4): SO4. The precipitate was taken up in a small volume of 1 mM benzarnidine solution, desalted by passage through Sephadex G-25, and lyophilized. The resulting powder (620 mg) contained less than 3% of nucleic acids. This powder (310 mg) was dissolved in 15 ml of a 50 mM Tris-HC1 buffer (pH 8.0) 1 mM in benzamidine and the solution was chromatographed on DEAEcellulose at pH 8.0 as indicated by Fig. lb. Elution of the column by a 0.0 to 0.4 M NaC1 gradient induced the separation of three peaks. The fractions of the first peak were found to hydrolyze N-acetyl-L-tyrosine ethylester after a 60-min incubation at pH 7.8 and 0 ° with trypsin (approximate enzyme-protein weight ratio, 1:50), and they were consequently assumed to contain some remaining procarboxypeptidase A. The second peak was not investigated. The fractions of the most anionic peak (90 mg after dialysis and lyophilization) displayed after incubation with enterokinase a strong activity towards benzoyl-L-arginine ethylester as well as a sizeable activity towards N-acetyl-L-tyrosine ethylester. Their nucleic acid content was found to be negligable by spectrophotometry. The top fraction of this peak gave a single band by disc electrophoresis (7.5 or 15% gel; pH 8.6). Using, by analogy with the cationic trypsinogen, a molar extinction coefficient value (Et~m)at 280 nm of 15.0 for the new zymogen, the specific activity of the fraction towards benzoyl-L.arginine ethylester after full activation by enterokinase was found to be about 40. This value compares favorably with the known potential activity of the pure cationic trypsinogen of bovine pancreas (about 50). The molecular weight of the reduced protein was determined by gel electrophoresis in the presence of sodium dodecyl sulfate and found to be 24 0 0 0 - 2 4 800. Similar assays with the cationic trypsinogen gave values ranging from 24 500 to 25 000. The new zymogen is certainly distinct from the cationic trypsinogen of bovine •kActivity appearing upon incubation of the zymogen with trypsin. Biochirn. Biophys. Acta, 236 (1971) 499-502
BBA REPORT
501
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20
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64 74 86 92
100 120 140
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150 No.
Fig. 1. Purification on DEAE-ceUulose of the anionic trypsinogen of bovine pancreas. a: First chromatography at pH 7.0. The 6 cm x 40 cm DEAE-cellulose (Brown, fibrous form) column was equilibrated with a 50 mM Tris-HCl buffer (pH 7.0) 1 mM in benzamidine. The diagram represents the emergence of the most anionic components of the extract after elution of the cationic and weakly anionic proteins (see text). Fraction volume, 28.5 ml. Elution rate, 300 ml/h. b: Fractions 31-86 in a were submitted to a second chromatography at pH 8.0 in a 2 cm x 40 cm DEAE-cellulose (Whatman, microgranular form) column equilibrated with a 50 mM Tris-HC1 buffgr (pH 8.0) 1 mM in benzamidine and eluted by a linear NaC1 gradient from 0.0 to 0.4 M. Fraction volume, 18 ml. Elution rate, 70 ml/h. ATEE, acetyl-L-tyrosineethylester. BAEE, benzoyl-L-arginine ethylester. ProCp A, procarboxypeptidase A. pancreas since the isoelectric points of the two proteins are widely different. Indeed, this zymogen appears to be the most anionic protein component of the investigated mixture, whereas the already known trypsinogen is strongly cationic. The dual activity of the corresponding enzyme towards benzoyl-L-arginine ethylester and N-acetyl-L-tyrosine ethylester is in agreement with the known action of the cationic trypsin on aromatic substrates 7,8 . Like the cationic trypsin, the anionic enzyme hydrolyzes tosyl-L-arginine methylester more rapidly than benzoyl-L-arginine ethylester and it readily activates chymotrypsinogen. Moreover, in sharp contrast with porcine kallikrein 9"11 , it has been observed to be fully inhibited, not only by the trypsin inhibitor of Kunitz, but also by the secretory pancreatic inhibitor of Kazal and by the soy bean inhibitor. This inhibition, which is highly specific for trypsin, confirms in an especially convincing way that the new zymogen isolated from bovine pancreas is a trypsinogen. One o f us (A.P.) is indebted to Drs. M. Rovery and S. Maroux for helpful discussions and useful advice.
Biochim. Biophys. Acta, 236 (1971) 499-502
502
BBA REPORT
REFERENCES 1 2 3 4 5 6 7 8 9 10 11
J.R. Brown, M. Yamasaki and H. Neurath, Biochemistry, 2 (1963) 867. R.J. Peanasky, D. Gratecos, J. Batatti and M. Rovery, Biochim. Biophys, Acta, 181 (1969) 82. J.P. Reboud, L. Pasero and P. Desnuelle, Biochem. Biophyg Reg Commun., 17 (1964) 347. P. Desnuelle, D. Gratecos, M. Charles, R. Peanasky, J. Batatti and M. Rovery, in P. Desnuelle, H. Neurath and M. Ottesen, Structure-Function Relationships in Proteolytic Enzymes, Munksgaard, Copenhagen, 1970, p. 21. C. Figarella, F. Clemente and O. Guy, FEBSLetters, 3 (1969) 351. S. Maroux, J. Baratti and P. Desnuelle, J. BioL Chem.,246 (1971) in the press. S. Matoux, M. Rovery and P. Desnuelle, Biochim. Biophyg Acta, 56 (1962) 202. S. Maroux, M. Rovery and P. Desnuelle, Biochim. Biophyg Acta, 122 (1966) 147. H. Fritz, F. Woitinas and E. Werle, Hoppe-Seyler's Z. PhysioL Chem., 345 (1966) 168. P.J. Burk, R.Z. HamiU, E.W. Cerwinsky and E.L. Gdnnan, Biochemistry, 6 (1967) 3180. E. Wetle and B. Kaufmann-Boetsch, Hoppe-Seyler'sZ. PhysioL Chem., 319 (196Q) 52.
Biochim. Biophys. Acta, 236 (1971) 499-502.
499
BBA R e p o r t BBA 31104 I d e n t i f i c a t i o n o f an a n i o n i c t r y p s i n o g e n in b o v i n e p a n c r e a s
A. PUIGSERVER and P. DESNUELLE Centre de Biochimie et de Biologic Moldculaire du ~N.R.S., 31, Chemin £ Aiguier 13, Marseille (France)
(Received March 8th, 1971)
SUMMARY A strongly anionic trypsinogen has been identified in bovine pancreas with the aid of enterokinase as the activating agent. The new zymogen has been substantially purified by two chromatographies on DEAE-cellulose.
The enzymes and zymogens of the exocrine secretion of bovine pancreas have received more attention than those of any other species. They are known to consist essentially of two chymotrypsinogens, a cationic trypsinogen, an amylase, a lipase, a deoxyribonuclease, a ribonuclease, a procarboxypeptidase B and a procarboxypeptidase A. This latter has been shown to be a trimer 1 composed of the zymogen of carboxypeptidase A, a proendopeptidase similar to porcine chymotrypsinogen C (ref. 2) and a still unidentified protein. The presence of a second trypsinogen in the bovine secretion has never been reported, although two trypsinogens have already been shown to exist in rat a , pig 4 and human s pancreas. The purification of the anionic procarboxypeptidase A of bovine pancreas was observed in this laboratory to be often associated with partial activation of the proendopeptidase monomer. This undesirable activation could not always be prevented by addition of 1 mM benzamidine and, therefore, it was not likely to be caused by traces of cationic trypsin possibly carried into the anionic protein fraction by some other components. An anionic bovine trypsinogen has now been identified with the aid of highly purified enterokinase 6 as the activating agent. According to the usual purification procedure for procarboxypeptidase A (ref. 1), an aqueous extract (1800 ml) of an acetone powder (180 g) of bovine pancreas was introduced into a 6 cm x 40 cm DEAE-ceUulose (Brown, fibrous form) column equilibrated with a 50 mM Tris-HCl buffer (pH 7.0) 1 mM in benzamidine. The cationic proteins were washed out by passage of 6.'5 1 of the buffer 0.06 M in NaC1, and a first portion of the anionic proteins was eluted using a linear gradient of NaCI from Biochim. Biophys. Acta, 236 (1971) 499-502
500
BBA REPORT
0.06 to 0.25 M. After the emergence of the peak containing procarboxypeptidase A (identified through its potential activity* towards N-acetyl-L-tyrosine ethylester), a second portion of anionic components (proteins and nucleic acids) was eluted by another gradient so ajusted as to raise the NaC1 molarity from 0.25 to 1.0 in a buffer volume of 41. The fractions of the ascending limb of the sharp peak thus obtained were found to display a strong activity towards the trypsin substrate benzoyl-L-arginine ethylester after a 1-h incubation with enterokinase (approximate enzyme-protein weight ratio, 1:100) in a 50 mM Tris-HC1 buffer (pH 7.8) 50 mM in CaCI2. These fractions were shown by disc electrophoresis (15% gel, pH 8.6) to contain several protein components. Their nucleic acid content was evaluated by spectrophotometry to be 40%. The result of a similar chromatography with a slower NaC1 gradient (0.25 to 0.50 M in 41 of buffer) is indicated in Fig. la. A number of fractions with potential activity towards benzoyl-L-arginine ethylester are seen to emerge just ahead of a broad peak for an approximate NaC1 molarity of 0.33. Disc electrophoresis showed that these fractions contained a major protein component and some impurities. The broad peak on the far right was probably composed of nucleic acids, since no detectable bands were visible in the corresponding electrophoresis diagrams. Fractions 3 1 - 8 6 in Fig. 1a were pooled and precipitated at 0 ° by 80% saturated (NH4): SO4. The precipitate was taken up in a small volume of 1 mM benzarnidine solution, desalted by passage through Sephadex G-25, and lyophilized. The resulting powder (620 mg) contained less than 3% of nucleic acids. This powder (310 mg) was dissolved in 15 ml of a 50 mM Tris-HC1 buffer (pH 8.0) 1 mM in benzamidine and the solution was chromatographed on DEAEcellulose at pH 8.0 as indicated by Fig. lb. Elution of the column by a 0.0 to 0.4 M NaC1 gradient induced the separation of three peaks. The fractions of the first peak were found to hydrolyze N-acetyl-L-tyrosine ethylester after a 60-min incubation at pH 7.8 and 0 ° with trypsin (approximate enzyme-protein weight ratio, 1:50), and they were consequently assumed to contain some remaining procarboxypeptidase A. The second peak was not investigated. The fractions of the most anionic peak (90 mg after dialysis and lyophilization) displayed after incubation with enterokinase a strong activity towards benzoyl-L-arginine ethylester as well as a sizeable activity towards N-acetyl-L-tyrosine ethylester. Their nucleic acid content was found to be negligable by spectrophotometry. The top fraction of this peak gave a single band by disc electrophoresis (7.5 or 15% gel; pH 8.6). Using, by analogy with the cationic trypsinogen, a molar extinction coefficient value (Et~m)at 280 nm of 15.0 for the new zymogen, the specific activity of the fraction towards benzoyl-L.arginine ethylester after full activation by enterokinase was found to be about 40. This value compares favorably with the known potential activity of the pure cationic trypsinogen of bovine pancreas (about 50). The molecular weight of the reduced protein was determined by gel electrophoresis in the presence of sodium dodecyl sulfate and found to be 24 0 0 0 - 2 4 800. Similar assays with the cationic trypsinogen gave values ranging from 24 500 to 25 000. The new zymogen is certainly distinct from the cationic trypsinogen of bovine •kActivity appearing upon incubation of the zymogen with trypsin. Biochirn. Biophys. Acta, 236 (1971) 499-502
BBA REPORT
501
roHltH
""1,
! v
20
E 0
40
64 74 86 92
100 120 140
a
,""
?0.6
io
;
o
J3 <
,,,
o., o.2t /
~;
~do Fraction
NO.
b
,~ ,,,
6-
ATEE
~
v
~4 o
;~,
~o
t,
',
4-
t'
/",,.I
2
~oo Fraction
i
150 No.
Fig. 1. Purification on DEAE-ceUulose of the anionic trypsinogen of bovine pancreas. a: First chromatography at pH 7.0. The 6 cm x 40 cm DEAE-cellulose (Brown, fibrous form) column was equilibrated with a 50 mM Tris-HCl buffer (pH 7.0) 1 mM in benzamidine. The diagram represents the emergence of the most anionic components of the extract after elution of the cationic and weakly anionic proteins (see text). Fraction volume, 28.5 ml. Elution rate, 300 ml/h. b: Fractions 31-86 in a were submitted to a second chromatography at pH 8.0 in a 2 cm x 40 cm DEAE-cellulose (Whatman, microgranular form) column equilibrated with a 50 mM Tris-HC1 buffgr (pH 8.0) 1 mM in benzamidine and eluted by a linear NaC1 gradient from 0.0 to 0.4 M. Fraction volume, 18 ml. Elution rate, 70 ml/h. ATEE, acetyl-L-tyrosineethylester. BAEE, benzoyl-L-arginine ethylester. ProCp A, procarboxypeptidase A. pancreas since the isoelectric points of the two proteins are widely different. Indeed, this zymogen appears to be the most anionic protein component of the investigated mixture, whereas the already known trypsinogen is strongly cationic. The dual activity of the corresponding enzyme towards benzoyl-L-arginine ethylester and N-acetyl-L-tyrosine ethylester is in agreement with the known action of the cationic trypsin on aromatic substrates 7,8 . Like the cationic trypsin, the anionic enzyme hydrolyzes tosyl-L-arginine methylester more rapidly than benzoyl-L-arginine ethylester and it readily activates chymotrypsinogen. Moreover, in sharp contrast with porcine kallikrein 9"11 , it has been observed to be fully inhibited, not only by the trypsin inhibitor of Kunitz, but also by the secretory pancreatic inhibitor of Kazal and by the soy bean inhibitor. This inhibition, which is highly specific for trypsin, confirms in an especially convincing way that the new zymogen isolated from bovine pancreas is a trypsinogen. One o f us (A.P.) is indebted to Drs. M. Rovery and S. Maroux for helpful discussions and useful advice.
Biochim. Biophys. Acta, 236 (1971) 499-502
502
BBA REPORT
REFERENCES 1 2 3 4 5 6 7 8 9 10 11
J.R. Brown, M. Yamasaki and H. Neurath, Biochemistry, 2 (1963) 867. R.J. Peanasky, D. Gratecos, J. Batatti and M. Rovery, Biochim. Biophys, Acta, 181 (1969) 82. J.P. Reboud, L. Pasero and P. Desnuelle, Biochem. Biophyg Reg Commun., 17 (1964) 347. P. Desnuelle, D. Gratecos, M. Charles, R. Peanasky, J. Batatti and M. Rovery, in P. Desnuelle, H. Neurath and M. Ottesen, Structure-Function Relationships in Proteolytic Enzymes, Munksgaard, Copenhagen, 1970, p. 21. C. Figarella, F. Clemente and O. Guy, FEBSLetters, 3 (1969) 351. S. Maroux, J. Baratti and P. Desnuelle, J. BioL Chem.,246 (1971) in the press. S. Matoux, M. Rovery and P. Desnuelle, Biochim. Biophyg Acta, 56 (1962) 202. S. Maroux, M. Rovery and P. Desnuelle, Biochim. Biophyg Acta, 122 (1966) 147. H. Fritz, F. Woitinas and E. Werle, Hoppe-Seyler's Z. PhysioL Chem., 345 (1966) 168. P.J. Burk, R.Z. HamiU, E.W. Cerwinsky and E.L. Gdnnan, Biochemistry, 6 (1967) 3180. E. Wetle and B. Kaufmann-Boetsch, Hoppe-Seyler'sZ. PhysioL Chem., 319 (196Q) 52.
Biochim. Biophys. Acta, 236 (1971) 499-502.