/ . Biochem. 84, 477-481 (1978)
Selective Cleavage of Peptide Bonds by a Serine Protease from Rat Skeletal Muscle1 Keiko KOBAYASHI,* Yukihiro SANADA, and Nobuhiko KATUNUMA1 Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Kuramoto-cho, Tokushima, Tokushima 770 Received for publication, April 18, 1978
The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr, Tyr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-GIu, were not susceptible to another serine protease from small intestine (Si-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the Si-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the Si-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the Si-protease.
ethyl ester, a substrate of trypsin (7). One of these enzymes, the Si-protease has been crystallized (7) and its selective cleavage of the peptide bonds in several polypeptides has been reported (70). Another of these intracellular serine proteases, the SK-protease has been purified to a homog1 This study was supported by a Grant-in-Aid for Scienti- eneous state (7) and it was recently crystallized by fic Research from the Ministry of Education, Science Sanada et al. (77). In the present study, we and Culture of Japan (No. 137017). investigated the proteolytic action of the SK• Present address: Department of Biochemistry, School protease using glucagon and neurotensin as subof Medicine, Yamagata University, Zao-Iida, Yamagata strates. The specificity of this enzyme was com990-23. pared with that of the Si-protease (70) and with * To whom reprint requests should be sent. Abbreviations: SK-protease, serine protease from that of bovine chymotrypsin A (12, 13). The skeletal muscle; ST-protease, serine protease from small results provide fundamental information on the mechanism of degradation of certain myofibrillar intestine. We have reported the purification and some properties of intracellular serine proteases from various organs of rats (7-9). These proteases hydrolyzed acetyl tyrosine ethyl ester, a substrate of chymotrypsin, but not/j-toluenesulfonyl arginine
Vol. 84, No. 2, 1978
477
K. KOBAYASHI, Y. SANADA, and N. KATUNUMA
478
proteins by this enzyme in vitro and in vivo. MATERIALS AND METHODS Materials—The crystalline serine protease from the skeletal muscle of Wistar strain rats was prepared by the method of Sanada et al. (77). The enzyme was dissolved in 0.25 M potassium phosphate buffer, pH 8.0. Other materials used in the present study were as described previously (70). Methods—Glucagon and neurotensin were dissolved in 0.01 M NaOH and distilled water, respectively, in a final concentration of 10 mg/ml. Glucagon (282 nmol in 1.0 ml of reaction mixture) was digested with 6.4 eg of the SK-protease at a molar ratio of substrate to enzyme of 1,000 :1 and 519 nmol of neurotensin in 1.0 ml of reaction mixture was digested with 16 pt% of the enzyme at a molar ratio of substrate to enzyme of 780 : 1. The molar concentration of the SK-protease was calculated assuming that its molecular weight is 24,000 (77). The total amounts of each substrate used were 15-20mg. The proteolytic hydrolysis was allowed to proceed at 37°C in 0.25 M potassium phosphate buffer, pH 8.0, and the reaction was followed by measuring increase in ar-amino groups with fluorescamine using the method of Duckworth et al. (74). No ar-amino groups were released on incubation of either polypeptide or protease alone. Values were calculated with Lleucine as a standard and expressed as mol of released ar-amino groups in leucine equivalents per mol of substrate polypeptide. After the reaction, the mixture was diluted, acetic acid was added to a final concentration of 5 %, and the mixture was rapidly frozen or applied to a Dowex 50W column. Fragments of polypeptides were separated by column chromatography on Dowex 50W (x8, H + form), equilibrated with 5% acetic acid. The column (25 cm x 2.0 cm) was eluted at a flow-rate of 25 ml/h under pressure and developed with a combination of stepwise and gradient increase of ammonium acetate buffer according to the method of Thompson (75) and Hirs et al. (16). As the ammonium ions had to be removed from the samples before the ninhydrin reaction, the eluted fractions were hydrolyzed with alkali. Then the products were subjected to the ninhydrin reaction (77). The absorption of the eluted fractions at
280 nm was also measured. The peptides isolated on Dowex 50W chromatography were lyophilized and dissolved in water. The methods used for determination of amino-terminal residues and amino acid analysis were as described previously (70). Peptides containing tryptophan were identified by their absorbance at 280 nm and with Ehrlich's reagent (/vdimethylaminobenzaldehyde) as described by Easley (18). RESULTS Figure 1 shows the time courses of hydrolysis of glucagon and neurotensin by the SK-protease. The extent of digestion at 37°C was measured by determining release of ar-amino groups with fluorescamine. Digestion was rapid at a molar ratio of glucagon to enzyme of 1,000 : 1 or of neurotensin to enzyme of 780 : 1, and a plateau was reached within 2 h, as shown in Fig. 1. Next the specificity of the hydrolysis was examined. Glucagon was hydrolyzed for 4 h at 37°C at a molar ratio of substrate to enzyme of 1,000 : 1, and then the reaction was stopped by adding a final concentration of 5% acetic acid and the mixture was applied to a Dowex 50W column. Five major components were separated and subjected to amino-terminal amino acid analysis by dansylation and to amino acid analysis. No
,-X
•*
DIGESTION
TIKE ( h
)
Fig. 1. Hydrolyses of glucagon and neurotensin by SK-protease. Hydrolysis was carried out at 37°C with 1 mg/ml of each substrate in 250 DIM potassium phosphate buffer, pH 8.0. Activity was determined by measuring release of a-amino groups with fluorescamine, as described in "MATERIALS AND METHODS." The molar ratios of substrate to enzyme were 1,000:1 (glucagon, • ) and 780:1 (neurotensin, x ) . / . Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
479
TABLE I. Amino acid compositions of fragments obtained by digestion of glucagon with SK-protease. Experimental conditions were as described in Fig. 1 and " MATERIALS AND METHODS." A total of 5.35 //mol of glucagon was digested. Values in parentheses are theoretical numbers of residues in the fragments. Fragment (molar ratio) Amino acid B
A
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Leucine Tyrosine
Phenylalanine Lysine Histidme
1.16 0.96 1.04 1.13
(1) (1)
0.98 (1) 1.12 (1) 1.00 (1)
C
0.99 (1)
(1)
» See text.
b
E
2.24 (2)
1. 01 (1) 1. 16 (1)
0.94 (1) 2.10 (2)
(1) 1. 01 (1) 1. 01 (1) 1. 06 (1)
0.90 (1)
0.69 (1) 1.13 (1)
0.98 (1)
0.98 (1)
1-01 (1) 1.03 (1)
0.74 (1) 1.61 (2)
Argirune Tryptophan Relative yield of fragment O^mol) N-Terrrunal amino acid Structure of fragment''
D
49.0% (2.62) His
1-6
54.8% (2.93) Thr 7-10
38.3% (2.05) Ser 11-13
39.6% (2.12) Leu 14-25
59.8% (3.20) Leu 26-29
Residue numbers in the glucagon.
fraction containing undigested intact glucagon was detected. The amino acid components, the aminoterminal amino acids and the relative yields of each fragment are given in Table I. The results show that the products were peptides containing residues 1-6 (fragment A), 7-10 (fragment B), 11-13 (fragment Q, 14-25 (fragment D), and 26-29 (fragment E), thus accounting for all the residues in glucagon. From these findings it was deduced that the enzyme mainly hydrolyzed the peptide bonds between phenylalanine (—6) and threonine (—7), between tyrosine (—10) and serine (—11), between tyrosine (—13) and leucine ( — 14), and between tryptophan (—25) and leucine (—26) in glucagon, as summarized in Fig. 2. Neurotensin was digested similarly for 4 h at 37°C at a molar ratio of 780 : 1 and the fragments were separated on a Dowex 50W column. Two Vol. 84, No. 2, 1978
main fragments were detected. No fraction containing undigested intact neurotensin was detected. As seen from the results of amino acid and the amino-terminal amino acid analyses in Table II, the products were peptides containing residues 1-11 (fragment A) and 12-13 (fragment B) of neurotensin, thus accounting for all the residues in neurotensin. No amino-terminal amino acid of fragment A was detectable because the pyrrolidone carboxyl residue is the original aminoterminal residue of neurotensin. Isoleucine was detected as the amino-terminal residue of fragment B. As summarized in Fig. 2, the SK-protease mainly hydrolyzed the bond between tyrosine ( — 11) and isoleucine ( — 12) in neurotensin. Figure 2 summarizes the peptide bonds in the two polypeptides cleaved by the SK-protease. These results were compared with those obtained
480
K. KOBAYASHI, Y. SANADA, and N. KATUNUMA
1 5 10 15 HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP
SKELETAL MUSCLE SERINE PROTEASE BOVINE CHYKOTRYPSIN A SHALL INTESTINE SERINE PROTEASE
20 25 ' -SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-6LN-TRP-LEU-MET-ASN-THR
HFIIROTFNSIN 1 5 10 (PYRO)GLU-LEU-TYR-GLU-ASN-LYS-PRO-ARG-ARG-PRO-TYR-ILE-LEU
SKELETAL MUSCLE SERINE PROTEASE
t
SMALL INTESTINE SERINE PROTEASE
Fig. 2. Summary of the specificity of SK-protease toward two polypeptides in comparison with the actions of bovine chymotrypsin A and Si-protease. Data on the action of chymotrypsin A on glucagon are from Bromer et al. (12) and Enenkel and Smillie (13). Data on the action of Si-protease on the glucagon and neurotensin are from Kobayashi and Katunuma (70). Large arrows indicate the major sites of action of the enzyme, and small arrows indicate other bonds cleaved by the enzyme under the individual experimental conditions employed.
TABLE II. Amino acid compositions of fragments obtained by digestion of neurotensin with SK-protease. Hydrolysis conditions were as described in Fig. 1 and " MATERIALS AND METHODS." A total of 8.3 l
Selective Cleavage of Peptide Bonds by a Serine Protease from Rat Skeletal Muscle1 Keiko KOBAYASHI,* Yukihiro SANADA, and Nobuhiko KATUNUMA1 Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Kuramoto-cho, Tokushima, Tokushima 770 Received for publication, April 18, 1978
The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr, Tyr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-GIu, were not susceptible to another serine protease from small intestine (Si-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the Si-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the Si-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the Si-protease.
ethyl ester, a substrate of trypsin (7). One of these enzymes, the Si-protease has been crystallized (7) and its selective cleavage of the peptide bonds in several polypeptides has been reported (70). Another of these intracellular serine proteases, the SK-protease has been purified to a homog1 This study was supported by a Grant-in-Aid for Scienti- eneous state (7) and it was recently crystallized by fic Research from the Ministry of Education, Science Sanada et al. (77). In the present study, we and Culture of Japan (No. 137017). investigated the proteolytic action of the SK• Present address: Department of Biochemistry, School protease using glucagon and neurotensin as subof Medicine, Yamagata University, Zao-Iida, Yamagata strates. The specificity of this enzyme was com990-23. pared with that of the Si-protease (70) and with * To whom reprint requests should be sent. Abbreviations: SK-protease, serine protease from that of bovine chymotrypsin A (12, 13). The skeletal muscle; ST-protease, serine protease from small results provide fundamental information on the mechanism of degradation of certain myofibrillar intestine. We have reported the purification and some properties of intracellular serine proteases from various organs of rats (7-9). These proteases hydrolyzed acetyl tyrosine ethyl ester, a substrate of chymotrypsin, but not/j-toluenesulfonyl arginine
Vol. 84, No. 2, 1978
477
K. KOBAYASHI, Y. SANADA, and N. KATUNUMA
478
proteins by this enzyme in vitro and in vivo. MATERIALS AND METHODS Materials—The crystalline serine protease from the skeletal muscle of Wistar strain rats was prepared by the method of Sanada et al. (77). The enzyme was dissolved in 0.25 M potassium phosphate buffer, pH 8.0. Other materials used in the present study were as described previously (70). Methods—Glucagon and neurotensin were dissolved in 0.01 M NaOH and distilled water, respectively, in a final concentration of 10 mg/ml. Glucagon (282 nmol in 1.0 ml of reaction mixture) was digested with 6.4 eg of the SK-protease at a molar ratio of substrate to enzyme of 1,000 :1 and 519 nmol of neurotensin in 1.0 ml of reaction mixture was digested with 16 pt% of the enzyme at a molar ratio of substrate to enzyme of 780 : 1. The molar concentration of the SK-protease was calculated assuming that its molecular weight is 24,000 (77). The total amounts of each substrate used were 15-20mg. The proteolytic hydrolysis was allowed to proceed at 37°C in 0.25 M potassium phosphate buffer, pH 8.0, and the reaction was followed by measuring increase in ar-amino groups with fluorescamine using the method of Duckworth et al. (74). No ar-amino groups were released on incubation of either polypeptide or protease alone. Values were calculated with Lleucine as a standard and expressed as mol of released ar-amino groups in leucine equivalents per mol of substrate polypeptide. After the reaction, the mixture was diluted, acetic acid was added to a final concentration of 5 %, and the mixture was rapidly frozen or applied to a Dowex 50W column. Fragments of polypeptides were separated by column chromatography on Dowex 50W (x8, H + form), equilibrated with 5% acetic acid. The column (25 cm x 2.0 cm) was eluted at a flow-rate of 25 ml/h under pressure and developed with a combination of stepwise and gradient increase of ammonium acetate buffer according to the method of Thompson (75) and Hirs et al. (16). As the ammonium ions had to be removed from the samples before the ninhydrin reaction, the eluted fractions were hydrolyzed with alkali. Then the products were subjected to the ninhydrin reaction (77). The absorption of the eluted fractions at
280 nm was also measured. The peptides isolated on Dowex 50W chromatography were lyophilized and dissolved in water. The methods used for determination of amino-terminal residues and amino acid analysis were as described previously (70). Peptides containing tryptophan were identified by their absorbance at 280 nm and with Ehrlich's reagent (/vdimethylaminobenzaldehyde) as described by Easley (18). RESULTS Figure 1 shows the time courses of hydrolysis of glucagon and neurotensin by the SK-protease. The extent of digestion at 37°C was measured by determining release of ar-amino groups with fluorescamine. Digestion was rapid at a molar ratio of glucagon to enzyme of 1,000 : 1 or of neurotensin to enzyme of 780 : 1, and a plateau was reached within 2 h, as shown in Fig. 1. Next the specificity of the hydrolysis was examined. Glucagon was hydrolyzed for 4 h at 37°C at a molar ratio of substrate to enzyme of 1,000 : 1, and then the reaction was stopped by adding a final concentration of 5% acetic acid and the mixture was applied to a Dowex 50W column. Five major components were separated and subjected to amino-terminal amino acid analysis by dansylation and to amino acid analysis. No
,-X
•*
DIGESTION
TIKE ( h
)
Fig. 1. Hydrolyses of glucagon and neurotensin by SK-protease. Hydrolysis was carried out at 37°C with 1 mg/ml of each substrate in 250 DIM potassium phosphate buffer, pH 8.0. Activity was determined by measuring release of a-amino groups with fluorescamine, as described in "MATERIALS AND METHODS." The molar ratios of substrate to enzyme were 1,000:1 (glucagon, • ) and 780:1 (neurotensin, x ) . / . Biochem.
SELECTIVE CLEAVAGE OF PEPTIDE BONDS BY A SERINE PROTEASE
479
TABLE I. Amino acid compositions of fragments obtained by digestion of glucagon with SK-protease. Experimental conditions were as described in Fig. 1 and " MATERIALS AND METHODS." A total of 5.35 //mol of glucagon was digested. Values in parentheses are theoretical numbers of residues in the fragments. Fragment (molar ratio) Amino acid B
A
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Leucine Tyrosine
Phenylalanine Lysine Histidme
1.16 0.96 1.04 1.13
(1) (1)
0.98 (1) 1.12 (1) 1.00 (1)
C
0.99 (1)
(1)
» See text.
b
E
2.24 (2)
1. 01 (1) 1. 16 (1)
0.94 (1) 2.10 (2)
(1) 1. 01 (1) 1. 01 (1) 1. 06 (1)
0.90 (1)
0.69 (1) 1.13 (1)
0.98 (1)
0.98 (1)
1-01 (1) 1.03 (1)
0.74 (1) 1.61 (2)
Argirune Tryptophan Relative yield of fragment O^mol) N-Terrrunal amino acid Structure of fragment''
D
49.0% (2.62) His
1-6
54.8% (2.93) Thr 7-10
38.3% (2.05) Ser 11-13
39.6% (2.12) Leu 14-25
59.8% (3.20) Leu 26-29
Residue numbers in the glucagon.
fraction containing undigested intact glucagon was detected. The amino acid components, the aminoterminal amino acids and the relative yields of each fragment are given in Table I. The results show that the products were peptides containing residues 1-6 (fragment A), 7-10 (fragment B), 11-13 (fragment Q, 14-25 (fragment D), and 26-29 (fragment E), thus accounting for all the residues in glucagon. From these findings it was deduced that the enzyme mainly hydrolyzed the peptide bonds between phenylalanine (—6) and threonine (—7), between tyrosine (—10) and serine (—11), between tyrosine (—13) and leucine ( — 14), and between tryptophan (—25) and leucine (—26) in glucagon, as summarized in Fig. 2. Neurotensin was digested similarly for 4 h at 37°C at a molar ratio of 780 : 1 and the fragments were separated on a Dowex 50W column. Two Vol. 84, No. 2, 1978
main fragments were detected. No fraction containing undigested intact neurotensin was detected. As seen from the results of amino acid and the amino-terminal amino acid analyses in Table II, the products were peptides containing residues 1-11 (fragment A) and 12-13 (fragment B) of neurotensin, thus accounting for all the residues in neurotensin. No amino-terminal amino acid of fragment A was detectable because the pyrrolidone carboxyl residue is the original aminoterminal residue of neurotensin. Isoleucine was detected as the amino-terminal residue of fragment B. As summarized in Fig. 2, the SK-protease mainly hydrolyzed the bond between tyrosine ( — 11) and isoleucine ( — 12) in neurotensin. Figure 2 summarizes the peptide bonds in the two polypeptides cleaved by the SK-protease. These results were compared with those obtained
480
K. KOBAYASHI, Y. SANADA, and N. KATUNUMA
1 5 10 15 HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP
SKELETAL MUSCLE SERINE PROTEASE BOVINE CHYKOTRYPSIN A SHALL INTESTINE SERINE PROTEASE
20 25 ' -SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-6LN-TRP-LEU-MET-ASN-THR
HFIIROTFNSIN 1 5 10 (PYRO)GLU-LEU-TYR-GLU-ASN-LYS-PRO-ARG-ARG-PRO-TYR-ILE-LEU
SKELETAL MUSCLE SERINE PROTEASE
t
SMALL INTESTINE SERINE PROTEASE
Fig. 2. Summary of the specificity of SK-protease toward two polypeptides in comparison with the actions of bovine chymotrypsin A and Si-protease. Data on the action of chymotrypsin A on glucagon are from Bromer et al. (12) and Enenkel and Smillie (13). Data on the action of Si-protease on the glucagon and neurotensin are from Kobayashi and Katunuma (70). Large arrows indicate the major sites of action of the enzyme, and small arrows indicate other bonds cleaved by the enzyme under the individual experimental conditions employed.
TABLE II. Amino acid compositions of fragments obtained by digestion of neurotensin with SK-protease. Hydrolysis conditions were as described in Fig. 1 and " MATERIALS AND METHODS." A total of 8.3 l
