/ . Biochem., 80, 659-669 (1976)
III. Hydrolysis of Peptides, and Inactivation of Angiotensin and Bradykinin by Cathepsin A Keiichi MATSUDA Central Research;Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140 Received for publication, April 23, 1976
Systematic analysis of the hydrolysis of benzyloxycarbonyl (Cbz)-dipeptides by cathepsin A [EC 3.4.12.1] purified from rat liver lysosomes showed that multiple forms of cathepsin A preferentially cleave peptide bonds with leucine, methionine, and phenylalanine. Cbz-Met-Met, -Met-Phe, -Phe-Met, and -Phe-Ala were hydrolyzed 6 to 8 times faster than the standard substrates, Cbz-Glu-Phe and Cbz-Glu-Tyr. The pH optima of the hydrolyses were 4.6 to 5.8. Hydrolysis of peptide bonds with glycine, isoleucine, and proline was very slow, but the rate depended on the nature of the adjacent amino acids. Proteins such as albumin, cytochrome c, j--globulin, hemoglobin, histone, myoglobin, and myosin were scarecely degraded. Peptide hormones, such as glucagon and adrenocorticotropic hormone (ACTH) were hydrolyzed markedly with optimum pH's of 4.5 and 4.6, respectively. Angiotensin I, II, bradykinin, Lys- and Met-Lysbradykinin (kallidin and Met-kallidin), and substance P were also hydrolyzed at appreciable rates. pH optima for these peptide hormones were 5.2 to 5.6. On the other hand, insulin and its A chain, luteinizing hormone-releasing hormone (LH-RH), oxytocin and vasopressin were cleaved slowly. In the hydrolyses of glucagon and other peptides, multiple forms of rat liver lysosomal cathepsin A again showed a carboxypeptidase nature, cleaving peptide bonds sequentially from the carboxyl terminal. Almost all of the amino acids were cleaved on prolonged incubation. Vaso-activities of angiotensin II and bradykinin were rapidly lost on hydrolysis by cathepsin A. Lysosomal cathepsin C [dipeptidylaminopeptidase I, EC 3.4.14.1] also inactivated angiotensin II, but did not inactivate bradykinin. Cathepsin A, therefore, can be regarded as one of the lysosomal angiotensinases and kininases. No distinct differences were observed between the multiple forms of cathepsin A in these hydrolyses and inactivations of peptides.
Abbreviations used : Cbz-, N»-benzyloxycarbonyl-(N«-carbobenzoxy-ar-); Nle, norleucine ; dansyl-, N-(l-dimethylaminonaphthalene-5-sulfonyl)-; ACTH, adrenocorticotropic hormone; LH-RH, luteinizing hormone-releasing hormone. Vol. 80, No. 4, 1976
659
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Studies on Cathepsins of Rat Liver Lysosomes
660
K. MATSUDA
Recently, the degradation of several peptide hormones, such as ACTH, angiotensin, and bradykinin by catheptic carboxypeptidases and dipeptidyl aminopeptidase I (cathepsin C) [EC 3.4.14.1] has been reported, and the possible participation of these exopeptidases in the inactivation of peptide hormones has been dis-
cussed (9, 11, 12). In the present report, the properties of multiple forms of rat liver lysosomal cathepsin A in hydrolyses of various Cbz-dipeptides, peptide hormones and proteins are described. Substrate specificities for synthetic peptides and peptide hormones are compared, and the possible roles of cathepsin A in the degradation of peptide hormones are discussed. MATERIALS AND METHODS Sources of Substrates and Reagents—CbzAsp-Gly, -Asp-Tyr, -Glu-Glu, -Glu-Leu, -GluMet, -Gly-Leu, -Gly-Lys, -His-Phe, -Met-Gly, -Met-Leu, -Met-Met, -Met-Phe, -Met-Pro, -NleLeu, -Nle-Nle, -Phe-Glu, and -Phe-Leu were obtained from Bachem Co., California. Cbz-AlaGly, -Glu-Gly, -Gly-Gly, -Gly-Tyr, -Leu-Phe, and -Nle-Phe were purchased from Cyclo
Chemical Co., Los Angeles. Cbz-Glu-Pro, -GlyPro, -Leu-Leu, -Leu-Met, -Phe-Gly, -Phe-Leu, -Phe-Met, -Phe-Pro, -Pro-Gly, -Pro-Leu, -ProMet, and -Pro-Phe were obtained from Fluka Chemische Fabrik, Buchs, Switzerland. CbzGlu-Phe, -Glu-Tyr, and -Gly-Phe were purchased from the Protein Research Foundation, Osaka. Cbz-Glu-Trp was obtained from Research Plus Laboratories Inc., Denville, New Jersey. Cbz-Ala-Phe, -Gly-Met, -Leu-Gly, -Leu-Ile, -Leu-Phe, -Ile-Gly, -Ile-Leu, -Ile-Ile, -He-Met, -Phe-Ala, dansyl chloride, and dansylamino acids were purchased from Sigma Co., St. Louis. Cbz - Leu - LeuOMe and Cbz-LeuPheOMe were prepared by Dr. A. Ito in our labolatory. Polyamide sheets were obtained from Seikagaku Kogyo, Tokyo. Angiotensin I, II, LH-RH, lysyl-bradykinin, methionyl-lysyl-bradykinin, oxytocin, substance P, and vasopressin were purchased from the Protein Research Foundation. Bovine glucagon was obtained from Calbiochem., California. Porcine ACTH, bradykinin, bovine pancreas insulin, potato acid phosphatase [EC 3.1.3.2], rabbit muscle aldolase [EC 4.1.2.13], E. coli asparaginase [EC 3.5.1.1], and glucokinase [EC 2.7.1.2], bovine liver /3-glucuronidase [EC 3.2.1.31], bovine pancreas ribonuclease [EC 3.1.4.22], and Jack bean urease [EC 3.5.1.5], bovine serum alubumin, horse heart cytochrome c, human /--globulin, calf thymus histone, and horse heart myoglobin were purchased from Sigma Co. A and B chains of insulin were prepared according to the method of Leach et al. (13). Dextranase [EC 3. 2.1.11] from Chaetomium gracile was supplied by Dr. J. Hattori of our laboratory, and rabbit muscle myosin was generous gift from Prof. Dr. S. Ebashi, University of Tokyo. Preparation of Cathepsins A and C— Multiple forms of cathepsin A and cathepsin C were purified from the lysosomal fraction of rat liver, as described in previous reports (2, 10). These enzymes were purified sufficiently to avoid significant contamination with other enzymes, especially other cathepsins and proteases. Enzyme Assay—The standard assay for cathepsin A was carried out with N°-benzyl/ . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/80/4/659/772904 by Stockholm University Library user on 22 January 2019
Cathepsin A [EC 3. 4.12.1] is one of the major forms of cathepsin present in the lysosomal fraction (7, 2), and the activity of the enzyme is greatly elevated in nutritional and hereditary muscular dystrophy (3, 4). This enzyme was originally considered to be a pepsinlike protease, because it splits the synthetic substrate of pepsin [EC 3.4.23.1], N°-benzyloxycarbonyl-L-glutamyl-tyrosine (Cbz-Glu-Tyr) (5). Recent studies have shown that it is a carboxypeptidase, and not a pepsin-like enzyme (6, 7). Several catheptic carboxypeptidases have been reported, each differing in substrate specificity, thiol and cofactor requirement (8, 9). However, the multiple forms of cathepsin A (Ai, An, Am) purified from rat liver lysosomes did not show any requirement for thiol or ions for activity, and were not affected by chelating agents which remove metal cofactors {10). Muscle cathepsin A showed little activity toward albumin, hemoglobin, or myoglobin, but a long polypeptide, glucagon, was hydrolyzed at an appreciable rate (3).
HYDROLYSIS OF PEPTIDES BY LYSOSOMAL CATHEPSIN A
Vol. 80, Np.. 4, 1976
matic amino acid analysis (Hitachi, KLH-5) according to the method of Moore and Stein {19). Asparagine, aspartic acid, glutamine, and glutamic acid were determined with and without asparaginase treatments by the method described by Iodice {6). Methioninamide and glycineamide of substance P and oxytocin were detected as methionine and glycine after incubation with 6N HC1 for 16 hr, at 90°. N-Terminal Analysis by the DansyI Method—The method of Gray and Hartley {20) was used to form highly fluorescent dansyl derivatives of amino acids released by the digestion of peptide hormones with cathepsin A. Dansylated amino acids were developed on polyamide thin-layer chromatography plates by the method of Woods and Wang {21). RESULTS Hydrolysis of Cbz-dipeptides—The substrate specificities of multiple forms of cathepsin A toward various Cbz-dipeptides were studied systematically. Rates of hydrolysis were compared at pH 5.8 using Cbz-Glu-Phe as the standard substrate for cathepsin A. Carboxylterminal leucine, methionine, phenylalanine, and tyrosine were cleaved comparatively rapidly (Table I). Peptide bonds with glycine were hydrolyzed at a lower rate, and bonds with proline were cleaved much slowly. Peptides containing methionine and phenylalanine were preferentially hydrolyzed (Table II). Cbz-MetMet, -Met-Phe, -Phe-Met, and -Phe-Ala were cleaved 6 to 8 times faster than Cbz-Glu-Phe. Rates of hydrolysis of leucine-containing Cbzdipeptides were 3 to 5 times that of Cbz-GluPhe (Table HI). • Amino acids at the carboxyl side of isoleucine were cleaved very slowly. Norleucine has a similar molecular structure to methionine, and peptide bonds with this synthetic amino acid were also cleaved rapidly. Methyl esters of carboxyl-terminal amino acids suppress the hydrolysis of their peptide bonds almost completely. pH-Activity Curves of Cathepsin A for Cbz-dipeptides—The multiple forms of lysosomal cathepsin A showed essentially the same pHactivity curves for Cbz-Glu-Phe; the optimal pH was 5.8, as reported previously (10). The
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oxycarbonyl-L-glutamyl-L-phenylalanine (CbzGlu-Phe) at 37° for 30 min, in 0.1 M sodium actate, pH 5.8, as reported previously {2). Hydrolyses of various Cbz-dipeptides were measured by essentially the same method. Hydrolyses of peptides and proteins were assayed at pH 5.0 in 0.1 M sodium acetate, at substrate concentrations of 0.5 mM and 0.25 mM, respectively, and the released amino acids were determined by the ninhydrin method. Aldolase and glucokinase were assayed according to the method described by Otto (14). Acid phosphatase and ^-glucuronidase were measured as reported previously (2). Dextranase was assayed by the method described by Janson and Porath {15). Urease was incubated with urea by the method of Sumner {16), and released ammonia was measured by the method of Seligson and Seligson {17). Ribonuclease was assayed by a slight modification of the method of Spahr and Hollingworth {18). One unit of enzyme activity is defined as that amount which produces 1 /imoles of reaction product per min. Inactivation of Angiotensin and Bradykinin by Cathepsins A and C — Angiotensin and bradykinin (0.2 mM) were incubated with cathepsin A (Ai, 81 milliunits, Am, 91 milliunits) in 200 p\ of 0.05 M sodium acetate, pH 5.0, and with cathepsin C (Cn, 121 milliunits), in 200 ft\ of 20 mM NaCl, 10 mM dithiothreitol, and 0.05 M sodium acetate, pH 6.0. After incubation at 37° for 20 or 60 min, the reaction mixture was frozen in dry ice-acetone and aliquots were injected intravenously into a rat to detect changes in vaso-activities. Recording of Blood Pressure—A male rat (Wistar-Imamichi, weighing 300 g) was anesthetized with urethane (1000 mg/kg, s.c). Arterial blood pressure was recorded using an electronic sphygmomanometer with a pressuredisplacement transducer (Ninon Kohden, MP4T). Amino Acid Analysis—Amino acids released from peptide hormones by cathepsin A were extracted with 5% trichloroacetic acid, and the acid was removed completely by shaking with ethyl acetate. The preparation was evaporated to dryness and subjected to auto-
661
662
K. MATSUDA TABLE II. Hydrolysis of Cbz-dipeptides containing methionine and phenylalanine by 'cathepsin A. Rates of hydrolysis are presented as a percentage of CbzGlu-Phe hydrolysis. The experimental conditions were the same as in Table I, except for the activities of cathepsin A (12-15 milliunits). Relative activity Substrate
Relative activity Substrate Ai
Cbz-Glu-Glu -Glu-Gly -Glu-Leu -Glu-Met , -Glu-Phe -Glu-Pro -Glu-Trp -Glu-Tyr Cbz-Gly-Glu -Gly-Gly -Gly-Leu
'
Cbz-Glu-Phe
103
105
100
100
2
L
5
2
104
• 105
0 5 144 119 100 1 1 102
1- • • • - • ' 2 4 • • 8
1 4
1
0
3 ''•
3
160
'
An
154
34"
38
' 43
-Gly-Lys
15
11
.13
-Gly-Met
42
49
46
-Gly-Phe
49 '
61
57
-Gly-Pro
1
0
0
-Gly-Tyr
11
6
11
Cbz-Asp-Gly
10
7
-Glu-Gly
3
3
5
-Ile-Gly
2
1
2
i:
-Leu-Gly
112
108
108
-Met-Gly
97
74
81
-Phe-Gly
225
217
252
Ai
An
Am
100
100
100
97
74
81
-Met-Leu'
460
476
540
-Met-Met
650
680
730
-Met-Phe
667.
754
780
-Met-Pro
22
23
33
Cbz-Glu-Met
103
105
-Gly-Met
42
49
. • 52
-lie-Met
32
30
26
-Leu-Met
355
388
383
-Met-Met
650
680
730
-Phe-Met
522
546
585
-Pro-Met
27
14
8
-Thr-Met
34
26
20
Cbz-Met-GIy
.
119
Cbz-Phe-Ala
740
760
770
-Phe-Glu
212
256
280
-Phe-Gly
225
217
252
-Phe-Leu
351
304
276
-Phe-Met
522
546
585
-Phe-Pro
29
30
35
Cbz-Ala-Phe
14
13
8
-Glu-Phe
100
100
100
Cbz-Pro-Gly
1
0
0
-Pro-Leu
22
17
12
-Pro-Met
27
14
8
-Pro-Phe
10
9
6
Cbz-Glu-Pro
2
1
1
-Lys-Phe
-Gly-Pro
1
0
0
-Met-Phe
-Leu-Pro
27
12
5
-Pro-Phe
10
-Met-Pro
22
23
33
-Phe-Pro
29
30
35
-Gly-Phe
49
62
52
-His-Phe
83
111
127
-Ile-Phe -Leu-Phe
4
9
16
294
308
394
108
140
109
667
754
780
9
6
/ . Biochtm.
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TABLE I. Hydrolysis of Cbz-dipeptides by cathepsin A. Cbz-dipeptides (10 mM) containing glutamine, glycine, and proline were incubated with multiple forms of cathepsin A (Ai, An, Am, 25-30 milliunits) in 0.1 M sodium acetate,. pH 5.8, at 37° for 30 min. Rates of hydrolysis are expressed as a percentage of Cbz-Glu-Phe hydrolysis.
663
HYDROLYSIS OF PEPTIDES BY LYSOSOMAL CATHEPSIN A
100
CbzL•u-1.au
CbzMat-Ph*
/
\
i?
/
Relative activity
50
Substrate
*
\
6.8
An Cbz-Glu-Phe
100
100
100
Cbz-Asp-Gly
10
7
11
-Asp-Tyr
46
40
45
Cbz-Leu-Gly
112
108
108
-Leu-Ile
222
181
176
-Leu-Leu
290
280
251
-Leu-Met
355
388
383
-Leu-Phe
294
308
394
.-Leu-Pro
27
12
5
0
1
-Leu-LeuOMe' -Lue-PheOMe Cbz-Glu-Leu
1 • 9
10
12
160
154
144
-Gly-Leu
34
38
43
-Ile-Leu
32
27
23
-Leu-Leu
290
280
251
-Met-Leu
460
476
540
-Phe-Leu
351
304
276
-Pro-Leu
44
17
12
Cbz-Ue-Gly -Ile-Ile
2
1
2
31
21
24
-Ile-Leu
32
27
23
-He-Met
32
30
26
-Ile-Phe
4
9
16
Cbz-Nle-Leu
131
140
156
-Nle-Nle
174
164
144
-Nle-Phe
242
206
175
pH-activity curves of preferentially hydrolyzed Cbz-dipeptides, Cbz-Leu-Leu, -Met-Met, -MetPhe, and -Phe-Ala, are shown in Fig. 1; their optimal pH's were 5.3, 5.2, 5.8, and 4.7, respectively. Hydrolysis of Proteins and Peptide Hormones—Relative rates of hydrolysis of polipeptides, proteins and peptide hormones by the multiple forms of cathepsin A were comVol. 80, No. 4, 1976
4
III. Hydrolysis of Peptides, and Inactivation of Angiotensin and Bradykinin by Cathepsin A Keiichi MATSUDA Central Research;Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140 Received for publication, April 23, 1976
Systematic analysis of the hydrolysis of benzyloxycarbonyl (Cbz)-dipeptides by cathepsin A [EC 3.4.12.1] purified from rat liver lysosomes showed that multiple forms of cathepsin A preferentially cleave peptide bonds with leucine, methionine, and phenylalanine. Cbz-Met-Met, -Met-Phe, -Phe-Met, and -Phe-Ala were hydrolyzed 6 to 8 times faster than the standard substrates, Cbz-Glu-Phe and Cbz-Glu-Tyr. The pH optima of the hydrolyses were 4.6 to 5.8. Hydrolysis of peptide bonds with glycine, isoleucine, and proline was very slow, but the rate depended on the nature of the adjacent amino acids. Proteins such as albumin, cytochrome c, j--globulin, hemoglobin, histone, myoglobin, and myosin were scarecely degraded. Peptide hormones, such as glucagon and adrenocorticotropic hormone (ACTH) were hydrolyzed markedly with optimum pH's of 4.5 and 4.6, respectively. Angiotensin I, II, bradykinin, Lys- and Met-Lysbradykinin (kallidin and Met-kallidin), and substance P were also hydrolyzed at appreciable rates. pH optima for these peptide hormones were 5.2 to 5.6. On the other hand, insulin and its A chain, luteinizing hormone-releasing hormone (LH-RH), oxytocin and vasopressin were cleaved slowly. In the hydrolyses of glucagon and other peptides, multiple forms of rat liver lysosomal cathepsin A again showed a carboxypeptidase nature, cleaving peptide bonds sequentially from the carboxyl terminal. Almost all of the amino acids were cleaved on prolonged incubation. Vaso-activities of angiotensin II and bradykinin were rapidly lost on hydrolysis by cathepsin A. Lysosomal cathepsin C [dipeptidylaminopeptidase I, EC 3.4.14.1] also inactivated angiotensin II, but did not inactivate bradykinin. Cathepsin A, therefore, can be regarded as one of the lysosomal angiotensinases and kininases. No distinct differences were observed between the multiple forms of cathepsin A in these hydrolyses and inactivations of peptides.
Abbreviations used : Cbz-, N»-benzyloxycarbonyl-(N«-carbobenzoxy-ar-); Nle, norleucine ; dansyl-, N-(l-dimethylaminonaphthalene-5-sulfonyl)-; ACTH, adrenocorticotropic hormone; LH-RH, luteinizing hormone-releasing hormone. Vol. 80, No. 4, 1976
659
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Studies on Cathepsins of Rat Liver Lysosomes
660
K. MATSUDA
Recently, the degradation of several peptide hormones, such as ACTH, angiotensin, and bradykinin by catheptic carboxypeptidases and dipeptidyl aminopeptidase I (cathepsin C) [EC 3.4.14.1] has been reported, and the possible participation of these exopeptidases in the inactivation of peptide hormones has been dis-
cussed (9, 11, 12). In the present report, the properties of multiple forms of rat liver lysosomal cathepsin A in hydrolyses of various Cbz-dipeptides, peptide hormones and proteins are described. Substrate specificities for synthetic peptides and peptide hormones are compared, and the possible roles of cathepsin A in the degradation of peptide hormones are discussed. MATERIALS AND METHODS Sources of Substrates and Reagents—CbzAsp-Gly, -Asp-Tyr, -Glu-Glu, -Glu-Leu, -GluMet, -Gly-Leu, -Gly-Lys, -His-Phe, -Met-Gly, -Met-Leu, -Met-Met, -Met-Phe, -Met-Pro, -NleLeu, -Nle-Nle, -Phe-Glu, and -Phe-Leu were obtained from Bachem Co., California. Cbz-AlaGly, -Glu-Gly, -Gly-Gly, -Gly-Tyr, -Leu-Phe, and -Nle-Phe were purchased from Cyclo
Chemical Co., Los Angeles. Cbz-Glu-Pro, -GlyPro, -Leu-Leu, -Leu-Met, -Phe-Gly, -Phe-Leu, -Phe-Met, -Phe-Pro, -Pro-Gly, -Pro-Leu, -ProMet, and -Pro-Phe were obtained from Fluka Chemische Fabrik, Buchs, Switzerland. CbzGlu-Phe, -Glu-Tyr, and -Gly-Phe were purchased from the Protein Research Foundation, Osaka. Cbz-Glu-Trp was obtained from Research Plus Laboratories Inc., Denville, New Jersey. Cbz-Ala-Phe, -Gly-Met, -Leu-Gly, -Leu-Ile, -Leu-Phe, -Ile-Gly, -Ile-Leu, -Ile-Ile, -He-Met, -Phe-Ala, dansyl chloride, and dansylamino acids were purchased from Sigma Co., St. Louis. Cbz - Leu - LeuOMe and Cbz-LeuPheOMe were prepared by Dr. A. Ito in our labolatory. Polyamide sheets were obtained from Seikagaku Kogyo, Tokyo. Angiotensin I, II, LH-RH, lysyl-bradykinin, methionyl-lysyl-bradykinin, oxytocin, substance P, and vasopressin were purchased from the Protein Research Foundation. Bovine glucagon was obtained from Calbiochem., California. Porcine ACTH, bradykinin, bovine pancreas insulin, potato acid phosphatase [EC 3.1.3.2], rabbit muscle aldolase [EC 4.1.2.13], E. coli asparaginase [EC 3.5.1.1], and glucokinase [EC 2.7.1.2], bovine liver /3-glucuronidase [EC 3.2.1.31], bovine pancreas ribonuclease [EC 3.1.4.22], and Jack bean urease [EC 3.5.1.5], bovine serum alubumin, horse heart cytochrome c, human /--globulin, calf thymus histone, and horse heart myoglobin were purchased from Sigma Co. A and B chains of insulin were prepared according to the method of Leach et al. (13). Dextranase [EC 3. 2.1.11] from Chaetomium gracile was supplied by Dr. J. Hattori of our laboratory, and rabbit muscle myosin was generous gift from Prof. Dr. S. Ebashi, University of Tokyo. Preparation of Cathepsins A and C— Multiple forms of cathepsin A and cathepsin C were purified from the lysosomal fraction of rat liver, as described in previous reports (2, 10). These enzymes were purified sufficiently to avoid significant contamination with other enzymes, especially other cathepsins and proteases. Enzyme Assay—The standard assay for cathepsin A was carried out with N°-benzyl/ . Biochem.
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Cathepsin A [EC 3. 4.12.1] is one of the major forms of cathepsin present in the lysosomal fraction (7, 2), and the activity of the enzyme is greatly elevated in nutritional and hereditary muscular dystrophy (3, 4). This enzyme was originally considered to be a pepsinlike protease, because it splits the synthetic substrate of pepsin [EC 3.4.23.1], N°-benzyloxycarbonyl-L-glutamyl-tyrosine (Cbz-Glu-Tyr) (5). Recent studies have shown that it is a carboxypeptidase, and not a pepsin-like enzyme (6, 7). Several catheptic carboxypeptidases have been reported, each differing in substrate specificity, thiol and cofactor requirement (8, 9). However, the multiple forms of cathepsin A (Ai, An, Am) purified from rat liver lysosomes did not show any requirement for thiol or ions for activity, and were not affected by chelating agents which remove metal cofactors {10). Muscle cathepsin A showed little activity toward albumin, hemoglobin, or myoglobin, but a long polypeptide, glucagon, was hydrolyzed at an appreciable rate (3).
HYDROLYSIS OF PEPTIDES BY LYSOSOMAL CATHEPSIN A
Vol. 80, Np.. 4, 1976
matic amino acid analysis (Hitachi, KLH-5) according to the method of Moore and Stein {19). Asparagine, aspartic acid, glutamine, and glutamic acid were determined with and without asparaginase treatments by the method described by Iodice {6). Methioninamide and glycineamide of substance P and oxytocin were detected as methionine and glycine after incubation with 6N HC1 for 16 hr, at 90°. N-Terminal Analysis by the DansyI Method—The method of Gray and Hartley {20) was used to form highly fluorescent dansyl derivatives of amino acids released by the digestion of peptide hormones with cathepsin A. Dansylated amino acids were developed on polyamide thin-layer chromatography plates by the method of Woods and Wang {21). RESULTS Hydrolysis of Cbz-dipeptides—The substrate specificities of multiple forms of cathepsin A toward various Cbz-dipeptides were studied systematically. Rates of hydrolysis were compared at pH 5.8 using Cbz-Glu-Phe as the standard substrate for cathepsin A. Carboxylterminal leucine, methionine, phenylalanine, and tyrosine were cleaved comparatively rapidly (Table I). Peptide bonds with glycine were hydrolyzed at a lower rate, and bonds with proline were cleaved much slowly. Peptides containing methionine and phenylalanine were preferentially hydrolyzed (Table II). Cbz-MetMet, -Met-Phe, -Phe-Met, and -Phe-Ala were cleaved 6 to 8 times faster than Cbz-Glu-Phe. Rates of hydrolysis of leucine-containing Cbzdipeptides were 3 to 5 times that of Cbz-GluPhe (Table HI). • Amino acids at the carboxyl side of isoleucine were cleaved very slowly. Norleucine has a similar molecular structure to methionine, and peptide bonds with this synthetic amino acid were also cleaved rapidly. Methyl esters of carboxyl-terminal amino acids suppress the hydrolysis of their peptide bonds almost completely. pH-Activity Curves of Cathepsin A for Cbz-dipeptides—The multiple forms of lysosomal cathepsin A showed essentially the same pHactivity curves for Cbz-Glu-Phe; the optimal pH was 5.8, as reported previously (10). The
Downloaded from https://academic.oup.com/jb/article-abstract/80/4/659/772904 by Stockholm University Library user on 22 January 2019
oxycarbonyl-L-glutamyl-L-phenylalanine (CbzGlu-Phe) at 37° for 30 min, in 0.1 M sodium actate, pH 5.8, as reported previously {2). Hydrolyses of various Cbz-dipeptides were measured by essentially the same method. Hydrolyses of peptides and proteins were assayed at pH 5.0 in 0.1 M sodium acetate, at substrate concentrations of 0.5 mM and 0.25 mM, respectively, and the released amino acids were determined by the ninhydrin method. Aldolase and glucokinase were assayed according to the method described by Otto (14). Acid phosphatase and ^-glucuronidase were measured as reported previously (2). Dextranase was assayed by the method described by Janson and Porath {15). Urease was incubated with urea by the method of Sumner {16), and released ammonia was measured by the method of Seligson and Seligson {17). Ribonuclease was assayed by a slight modification of the method of Spahr and Hollingworth {18). One unit of enzyme activity is defined as that amount which produces 1 /imoles of reaction product per min. Inactivation of Angiotensin and Bradykinin by Cathepsins A and C — Angiotensin and bradykinin (0.2 mM) were incubated with cathepsin A (Ai, 81 milliunits, Am, 91 milliunits) in 200 p\ of 0.05 M sodium acetate, pH 5.0, and with cathepsin C (Cn, 121 milliunits), in 200 ft\ of 20 mM NaCl, 10 mM dithiothreitol, and 0.05 M sodium acetate, pH 6.0. After incubation at 37° for 20 or 60 min, the reaction mixture was frozen in dry ice-acetone and aliquots were injected intravenously into a rat to detect changes in vaso-activities. Recording of Blood Pressure—A male rat (Wistar-Imamichi, weighing 300 g) was anesthetized with urethane (1000 mg/kg, s.c). Arterial blood pressure was recorded using an electronic sphygmomanometer with a pressuredisplacement transducer (Ninon Kohden, MP4T). Amino Acid Analysis—Amino acids released from peptide hormones by cathepsin A were extracted with 5% trichloroacetic acid, and the acid was removed completely by shaking with ethyl acetate. The preparation was evaporated to dryness and subjected to auto-
661
662
K. MATSUDA TABLE II. Hydrolysis of Cbz-dipeptides containing methionine and phenylalanine by 'cathepsin A. Rates of hydrolysis are presented as a percentage of CbzGlu-Phe hydrolysis. The experimental conditions were the same as in Table I, except for the activities of cathepsin A (12-15 milliunits). Relative activity Substrate
Relative activity Substrate Ai
Cbz-Glu-Glu -Glu-Gly -Glu-Leu -Glu-Met , -Glu-Phe -Glu-Pro -Glu-Trp -Glu-Tyr Cbz-Gly-Glu -Gly-Gly -Gly-Leu
'
Cbz-Glu-Phe
103
105
100
100
2
L
5
2
104
• 105
0 5 144 119 100 1 1 102
1- • • • - • ' 2 4 • • 8
1 4
1
0
3 ''•
3
160
'
An
154
34"
38
' 43
-Gly-Lys
15
11
.13
-Gly-Met
42
49
46
-Gly-Phe
49 '
61
57
-Gly-Pro
1
0
0
-Gly-Tyr
11
6
11
Cbz-Asp-Gly
10
7
-Glu-Gly
3
3
5
-Ile-Gly
2
1
2
i:
-Leu-Gly
112
108
108
-Met-Gly
97
74
81
-Phe-Gly
225
217
252
Ai
An
Am
100
100
100
97
74
81
-Met-Leu'
460
476
540
-Met-Met
650
680
730
-Met-Phe
667.
754
780
-Met-Pro
22
23
33
Cbz-Glu-Met
103
105
-Gly-Met
42
49
. • 52
-lie-Met
32
30
26
-Leu-Met
355
388
383
-Met-Met
650
680
730
-Phe-Met
522
546
585
-Pro-Met
27
14
8
-Thr-Met
34
26
20
Cbz-Met-GIy
.
119
Cbz-Phe-Ala
740
760
770
-Phe-Glu
212
256
280
-Phe-Gly
225
217
252
-Phe-Leu
351
304
276
-Phe-Met
522
546
585
-Phe-Pro
29
30
35
Cbz-Ala-Phe
14
13
8
-Glu-Phe
100
100
100
Cbz-Pro-Gly
1
0
0
-Pro-Leu
22
17
12
-Pro-Met
27
14
8
-Pro-Phe
10
9
6
Cbz-Glu-Pro
2
1
1
-Lys-Phe
-Gly-Pro
1
0
0
-Met-Phe
-Leu-Pro
27
12
5
-Pro-Phe
10
-Met-Pro
22
23
33
-Phe-Pro
29
30
35
-Gly-Phe
49
62
52
-His-Phe
83
111
127
-Ile-Phe -Leu-Phe
4
9
16
294
308
394
108
140
109
667
754
780
9
6
/ . Biochtm.
Downloaded from https://academic.oup.com/jb/article-abstract/80/4/659/772904 by Stockholm University Library user on 22 January 2019
TABLE I. Hydrolysis of Cbz-dipeptides by cathepsin A. Cbz-dipeptides (10 mM) containing glutamine, glycine, and proline were incubated with multiple forms of cathepsin A (Ai, An, Am, 25-30 milliunits) in 0.1 M sodium acetate,. pH 5.8, at 37° for 30 min. Rates of hydrolysis are expressed as a percentage of Cbz-Glu-Phe hydrolysis.
663
HYDROLYSIS OF PEPTIDES BY LYSOSOMAL CATHEPSIN A
100
CbzL•u-1.au
CbzMat-Ph*
/
\
i?
/
Relative activity
50
Substrate
*
\
6.8
An Cbz-Glu-Phe
100
100
100
Cbz-Asp-Gly
10
7
11
-Asp-Tyr
46
40
45
Cbz-Leu-Gly
112
108
108
-Leu-Ile
222
181
176
-Leu-Leu
290
280
251
-Leu-Met
355
388
383
-Leu-Phe
294
308
394
.-Leu-Pro
27
12
5
0
1
-Leu-LeuOMe' -Lue-PheOMe Cbz-Glu-Leu
1 • 9
10
12
160
154
144
-Gly-Leu
34
38
43
-Ile-Leu
32
27
23
-Leu-Leu
290
280
251
-Met-Leu
460
476
540
-Phe-Leu
351
304
276
-Pro-Leu
44
17
12
Cbz-Ue-Gly -Ile-Ile
2
1
2
31
21
24
-Ile-Leu
32
27
23
-He-Met
32
30
26
-Ile-Phe
4
9
16
Cbz-Nle-Leu
131
140
156
-Nle-Nle
174
164
144
-Nle-Phe
242
206
175
pH-activity curves of preferentially hydrolyzed Cbz-dipeptides, Cbz-Leu-Leu, -Met-Met, -MetPhe, and -Phe-Ala, are shown in Fig. 1; their optimal pH's were 5.3, 5.2, 5.8, and 4.7, respectively. Hydrolysis of Proteins and Peptide Hormones—Relative rates of hydrolysis of polipeptides, proteins and peptide hormones by the multiple forms of cathepsin A were comVol. 80, No. 4, 1976
4
