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Comparison of the elastolytic effects of human leukocyte elastase and porcine pancreatic elastase
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
COMPARISON OF THE ELASTOLYTIC EFFECTS OF HUMAN LEUKOCYTE ELASTASE AND PORCINE PANCREATIC ELASTASE Robert M. S e n i o r , David R. B i e l e f e l d and Barry C .
Starcher
Pulmonary D i v i s i o n , D e p a r t m e n t of M e d i c i n e , W a s h i n g t o n U n i v e r s i t y S c h o o l of M e d i c i n e at The I e w i s h H o s p i t a l of St. L o u i s , St. L o u i s , M i s s o u r i 63110 Received August
18,1976
S U M M A R Y : Porcine and bovine elastins were digested by h u m a n leukocyte elastase and porcine pancreatic elastase. The enzymes showed similarities in the extent to which they digested elastin and the pattern and quantitative distribution of N-termi nal amino acids in the digests. However, fingerprints of the digests showed differences between the products of leukocyte elastase and pancreatic elastase. Each enzyme produced its characteristic fingerprint irrespective of whether the elastin substrate was obtained from ligament, pleura or lung parenchyma. The enzymes also digested tropoelastin differently. The results suggest that leukocyte elastase and pancreatic elastase should not be considered interchangeable in experimental models of tissue injury. INTRODUCTION In 1968 [anoff and Scherer reported that granules of human leukocytes contain elastolytic activity(1) . In contrast to the previously identified pancreatic elastase, leukocyte elastase better withstood high ionic strength and acidic conditions.
Leukocyte elastase and pancreatic elastase responded
differently to inhibitOrs. Soybean trypsin inhibitor and salivary kallikrein inhibitor blocked the activity of leukocyte elastase but not of pancreatic elastase. O n the other hand, leukocyte elastase was less effectively inhibited by h u m a n serum than was pancreatic elastase. Subsequently, leukocyte elastase has been purified and other differences have been observed between it and pancreatic elastase.
Leukocyte elastase
appears to exist in multiple forms with different carbohydrate contents, but with identical amino acid compositions (2-5) . The amino acid composition of leukocyte elastase differs from that of pancreatic elastase.
Copyright © 1976 by Academic Press, Inc. All rights o] reproduction in any form reserved.
1327
Preliminary data on the amino-
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
terminal sequences indicate differences between leukocyte elastase and pancreatic elastase (6) . Immunological studies also suggest dlssimilarity(6) . Recently it has been shown that leukocyte elastase appears less active on a molar basis than pancreatic elastase against ligament elastin (6 ,7). The above differences between leukocyte elastase and pancreatic elastase suggest that these enzymes may display qualitative differences in the manner in which they digest elastin. It is possible therefore that features of tissue injury due to leukocyte elastase might not be accurately reflected by studies using pancreatic elastase.
The present study was undertaken to
compare the peptide bond specificities of leukocyte elastase and pancreatic elastase during the catalysis of elastin hydrolysis.
MATERIALS AND METHODS Elastin: Most studies were done with elastin purified from beef neck ligament(8). For studies using porcine pleural elastin, porcine lung parenchymal elastin and porcine tropoelastin the substrates were provided by Dr. L. B. Sandberg (9 'I0). Elastase: Chromatographically pure pancreatic elastase was purchased from Worthington Biochemical Corp., Freehold, N.J. and from Elastin Products, St.Louis, M o . Leukocyte elastase was purified by the method of Baugh and Tr~is from leukocytes collected by leukapheresis from one healthy male age 39'~°1. The enzyme was stored at -70°C after lyophilization. Under these conditions the enzyme retained activity indefinitely. The quantities of enzymes used were determined from extinction coefficients after measuring optical density at 280nM. Fingerprinting of elastin digests: Four milligrams of elastin or tropoelastin were incubated at 37°C with elastase in 0.1 M a m m o n i u m blcarbonate, 0.1 M a m m o n i u m acetate, p H 8.0. The ratios of enzyme to substrate were i:i00 and 1:50 for pancreatic elastase and leukocyte elastase respectively. After two hours an equivalent amount of fresh enzyme was added to the leukocyte elastase reaction mixture. Complete solubilization of substrate occurred after four hours. The reactions were terminated by addition of diisopropylflurophosphate to a final concentration of 5 m M . Two milligrams of solubilized elastin was suspended in 50% pyridine and spotted on W h a t m a n 3 M M paper. Chromatography, followed by high voltage electrophoresis, was performed as described by Sandberg et al (II) . Estimation of the size distribution and the N-terminal amino acids of the peptides in the e l a s t i n d i g e s t s : Fifty milligrams of ligament e l a s t i n was suspended in 1 ml of 0.05 M Tris-HC1 (pH 8.2) and incubated with 100 ug of l e u k o c y t e e l a s t a s e or p a n c r e a t i c e l a s t a s e at 37°C for 24 hours. Eight milligrams of the e l a s t i n d i g e s t s were d i a l y z e d s e p a r a t e l y in 6000-8000 or 12000-14000 molecular weight cutoff membranes (Spectrapor, Spectrum M e d i c a l I n d u s t r i e s , Los Angeles, CA) a g a i n s t 3 c h a n g e s of 50ml of water. The d i a l y s a t e s and the c o n t e n t s of the d i a l y s i s bags
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were evaporated to dryness. Each fraction then underwent hydrolysis and amino acid analysis as previously described(8). To determine the N-terminal amino acids 100ul of elastin digest (5mg) was mixed with 200ul of 0.1 M N a H C O 3 , 100ul dansyl chloride (Img/ml in acetone) and 10ul (10uCi) tritiated dansylchloride (New England Nuclear). The reaction mixture was allowed to dansylate at room temperature for 24 hours. The contents were evaporated employing a flow of nitrogen gas and then hydrolysed in screw top vials using 200ul of 6N HCI at 95°C for 12 hours. The contents were evaporated again and then dissolved in 20ul acetone - glacial acetic acid (4:1 ,v/v). One microliter of the mixture was used for thin layer chromatography on polyamide as described by W o o d s and W a n g (12) . The identity of the dansylated amino acids was established by control chromatograms made with mixtures of pure dansylated amino acids (Sigma Chemical Corp., St. Louis, MO). To quantitate the dansylated amino acids the dansyl spots were cut out, suspended in scintillation fluid and measured for [3H ] activity. RESULTS Chromatograms of digests of elastin and tropoelastin showed differences between the products of pancreatic elastase and leukocyte elastase (Figure i). A prominent component present near the origin in leukocyte elastase digests was not present in the pancreatic elastase digests.
Conversely, the pancreatic
elastase digests had intensely stained zones near the center of the chromatograms which were not seen in the chromatograms of substrates digested by leukocyte elastase. For each enzyme the chromatographic pattern was consistent irrespective of whether the elastin was derived from ligament, lung parenchyma or pleura of bovine or porcine origin. Fingerprints confirmed that leukocyte elastase and pancreatic elastase produce different peptides from elastin and tropoelastin (Figure 2). The prominent zone near the origin on the chromatograms of leukocyte elastase digests was not a single spot after high voltage electrophoresis.
Instead it was dispersed into
many spots along the length of the electrophoretogram which were very faint in elastin digests, but easily seen in tropoelastln digests. In contrast, the heavily stained zone observed near the center of the chromatogram of pancreatic elastase digests appeared to correspond to a single spot after electrophoresis.
1329
Similar
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,
Figure 1 Chromatograms of bovine ligament e l a s t i n d i g e s t e d by leukocyte e l a s t a s e (1) and p a n c r e a t i c e l a s t a s e (2) and t r o p o e l a s t i n digested by l e u k o c y t e e l a s t a s e (3) and p a n c r e a t i c e l a s t a s e (4). The arrow i n d i c a t e s the origin and direction of chromatography. At the top is shown the s e p a r a t i o n of a mixture of amino a c i d s .
to the r e s u l t s with c h r o m a t o g r a p h y , e a c h enzyme produced its d i s t i n c t i v e fingerprint independent of the s o u r c e of the e l a s t i n s u b s t r a t e . D i a l y s i s experiments demonstrated that l e u k o c y t e e l a s t a s e and p a n c r e a t i c e l a s t a s e were similar in digesting a large p e r c e n t a g e of e l a s t i n into peptides with molecular weights below 14000 (Table 1).
The e n z y m e s a l s o resembled one
another in that d e s m o s i n e s were only slightly r e p r e s e n t e d in peptides smaller
1330
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
+
,+,
,+,y+
+:+,r
+,++
-
+,! + +,
+~,
tI
,
)
2 Figure 2 Fingerprints of bovine ligament elastln digested by pancreatic elastase (I) and leukoctye elastase (2) and tropoelastin digested by pancreatic elastase (3) and leukocyte elastase (4). Chromatography was run in the vertical direction from top to bottom; electrophoresis was run horizontally. Amino acid standards, seen at the bottom of (2) and (4), were included in the electrophoreses to evaluate relative migration.
than 8000 molecular weight, but greater than 50 per cent of the desmoslnes were constituents of peptides in the range 8000-14000 molecular weight. The N-terminal amino acids did not appear to specify the peptide bonds of elastin broken by pancreatic elastase and leukocyte elastase during the hydrolysis of elastin. The N-terminal amino acids and their relative quantities
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TABLE 1 C O M P A R I S O N OF THE DIALYSABLE C O M P O N E N T S OF LIGAMENT ELASTIN AFTER DIGESTION BY PANCREATIC ELASTASE A N D LEUKOCYTE ELASTASE D i a l y s i s (molecular weight range)
6000 -
D i a l y s a b l e weight (per cent of the weight of the t o t a l digest)
Dialysable desmosines (per cent of the d e s m o s i n e s in the t o t a l digest)
Pancreatic Elastase Diqest
Pancreatic Elastase Dige st
Leukocyte Elastase Dige st
Leukocyte Elastase Digest
8000
44
41
Trace
Trace
12000 - 14000
83
76
58
53
TABLE 2 N-TERMINAL A M I N O ACIDS OF ELASTIN DIGESTS* N-Terminal Amino Acids
Elastin Digested By P~ncreatic E l a s t a s e [~H] (cpm)
Elastin Digested By Leukocyte E l a s t a s e [~H] (cpm)
Glycine
31,146
28,775
Alanine
21,250
16,426
Phenylalanine
4,490
4,155
Va line
3,560
4,006
Leucine
2,654
3,061
Proline
1,491
2,093
Isoleucine
1,539
912
*Equal amounts of r a d i o l a b e l e d , d a n s y l a t e d e l a s t i n d i g e s t were s u b j e c t e d to chromatography.
were similar for the peptides of e l a s t i n resulting from digesting by either enzyme (Table 2).
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Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS DISCUSSION
The p r e s e n t s t u d y d e m o n s t r a t e s t h a t t h e p e p t i d e b o n d s p e c i f i c i t y of leukocyte elastase
on elastin and tropoelastin substrates
pancreatic elastase.
d i f f e r s from t h a t o f
These differences have been demonstrated in chromatograms
a n d f i n g e r p r i n t s of d i g e s t s p r o d u c e d b y t h e t w o e n z y m e s .
That these enzymes
might attack elastin differently is not surprising since other differences between them are known.
It i s of c o n s i d e r a b l e
interest,
however, that mammals should
h a v e t w o a v e n u e s for t h e d i g e s t i o n of s u c h a n u n u s u a l p r o t e i n a s e l a s t i n . The d i s t i n c t i v e f i n g e r p r i n t p a t t e r n s t h a t r e s u l t e d from e l a s t o l y s i s leukocyte elastase the elastin.
and pancreatic elastase
by
w e r e i n d e p e n d e n t of t h e s o u r c e of
E a c h e n z y m e p r o d u c e d a c o n s i s t e n t f i n g e r p r i n t on r e a c t i o n w i t h
e l a s t i n s from s e v e r a l t i s s u e s
from p i g a n d c o w .
These data should not be inter-
p r e t e d t o m e a n t h a t e l a s t i n s a r e i d e n t i c a l in a l l m a m m a l i a n t i s s u e s .
Indeed,
d i f f e r e n c e s in e l a s t i n s a r e k n o w n to e x i s t b e t w e e n s p e c i e s (8) a n d a n t i b o d i e s t o e l a s t i n h a v e b e e n d e v e l o p e d in a n i m a l s (13) . On t h e o t h e r h a n d , t h e f i n g e r p r i n t d a t a s u g g e s t t h a t m a j o r d i f f e r e n c e s i n e l a s t i n s t r u c t u r e from t i s s u e to t i s s u e and between species are unlikely. Although leukocyte elastase is nevertheless
a true elastase.
d i f f e r s from p a n c r e a t i c e l a s t a s e , It d i g e s t e d t h e e l a s t i n
the enzyme
u s e d in t h e s e e x p e r i m e n t s
t o a n e x t e n t s i m i l a r to t h a t a c h i e v e d b y p a n c r e a t i c e l a s t a s e
and it h a s b e e n
s h o w n c a p a b l e o f d i g e s t i n g a v a r i e t y of e l a s t i n s l a b e l e d w i t h c h r o m o g e n s or radioisotopes(1,3,7).
Moreover, incubations of leukocyte elastase
with tissues
p r o d u c e h i s t o l o g i c e v i d e n c e o f t h e b r e a k d o w n of e l a s t i c f i b e r s (14) . L e u k o c y t e elastase
is inhibited by active site directed chloromethylketones which
s p e c i f i c a l l y i n h i b i t p a n c r e a t i c e l a s t a s e (15) . Pancreatic elastase of elastolysis
h a s b e e n u s e d a s a m o d e l of t h e p o s s i b l e e f f e c t s
upon tissues,
n o t a b l y in e x p e r i m e n t a l e m p h y s e m a ( 1 6 ) . It s e e m s
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much more l i k e l y , however, that l e u k o c y t e e l a s t a s e , rather than p a n c r e a t i c e l a s t a s e , may initiate the p a t h o g e n e s i s of d i s e a s e p r o c e s s e s such as pulmonary emphysema and joint damage (17) . The p r e s e n t study i n d i c a t e s that p a n c r e a t i c e l a s t a s e and l e u k o c y t e e l a s t a s e have different e f f e c t s a g a i n s t e l a s t i n .
Thus,
c a u t i o n should be e x e r c i s e d in the extrapolation of experimental r e s u l t s from p a n c r e a t i c e l a s t a s e to t h o s e r e s u l t s which might o c c u r with l e u k o c y t e e l a s t a s e .
Acknowledgments This work was supported by U S P H S Grant HL 16118. The authors gratefully acknowledge Dr. L. B. Sandberg for elastin and tropoelastin samples and for assistance in fingerprinting.
Reference s i. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17.
Janoff,A. and Scherer, I. (1968) I. Exp. Med. 128:1137-I151. lanoff,A. (1973) Lab. Invest. 2_~9:458-464. Ohlsson, K. and Olsson, I. (1974) Eur. J. Biochem. 4_~2:519-527. Taylor, J.C. and Crawford, I.P. (1975) Arch. Biochem. and Biophys. 169:91-101. Feinstein, G. and lanoff, A. (1975) Biochem. Biophys. Acta 403: 493-505. Baugh, R.J. and Travls, I. (1976) Biochem. i_~5:836-841. Bielefeld,D.R., Senior, R.M. and Yu, S.Y. (1975) Biochem. Biophys. Res. C o m m . 6_/7:1553-1559. Starcher, B.C. and Galione, M.I. (1976) Analytical Biochem., In Press. Rasmussen, B.L., Bruenger, E. and Sandberg, L.B. (1975) Analytical Biochem. 6._44:255-259. Sandberg, L.B., Bruenger, E. and Cleary, E.G. (1975) Analytical Biochem., 6_~4:249-254. Sandberg, L.B., Weissman, N. and Gray, W.R. (1971) Biochem. 1_O0:52-56. W o o d s , K.R. and Wang, K.T. (1967) Biochem. Biophys. Acta 133:369-370. S y k e s , B . C . and Partridge, S . M . (1974) Biochem. J. 14___~1:567-572. Janoff, A. (1970) Lab. I n v e s t . 2__2:228-235. Tuhy, P . M . and Powers, J . C . (1975) FEBS LETTERS 5 0 : 3 5 9 - 3 6 1 . Kaplan, P . D . , Kuhn, C . , and Pierce, J.A. (1973) I. Lab. C l i n . Meal. 82:349-356. lanoff, A . , Blondin, I . , Sandhaus, R . A . , M o s s e r , A . , and Malemud, C. (1975) Proteases and Biological Control, Cold Spring Harbor Conf. Cell Proliferation 2 : 5 0 3 - 6 2 0 .
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COMPARISON OF THE ELASTOLYTIC EFFECTS OF HUMAN LEUKOCYTE ELASTASE AND PORCINE PANCREATIC ELASTASE Robert M. S e n i o r , David R. B i e l e f e l d and Barry C .
Starcher
Pulmonary D i v i s i o n , D e p a r t m e n t of M e d i c i n e , W a s h i n g t o n U n i v e r s i t y S c h o o l of M e d i c i n e at The I e w i s h H o s p i t a l of St. L o u i s , St. L o u i s , M i s s o u r i 63110 Received August
18,1976
S U M M A R Y : Porcine and bovine elastins were digested by h u m a n leukocyte elastase and porcine pancreatic elastase. The enzymes showed similarities in the extent to which they digested elastin and the pattern and quantitative distribution of N-termi nal amino acids in the digests. However, fingerprints of the digests showed differences between the products of leukocyte elastase and pancreatic elastase. Each enzyme produced its characteristic fingerprint irrespective of whether the elastin substrate was obtained from ligament, pleura or lung parenchyma. The enzymes also digested tropoelastin differently. The results suggest that leukocyte elastase and pancreatic elastase should not be considered interchangeable in experimental models of tissue injury. INTRODUCTION In 1968 [anoff and Scherer reported that granules of human leukocytes contain elastolytic activity(1) . In contrast to the previously identified pancreatic elastase, leukocyte elastase better withstood high ionic strength and acidic conditions.
Leukocyte elastase and pancreatic elastase responded
differently to inhibitOrs. Soybean trypsin inhibitor and salivary kallikrein inhibitor blocked the activity of leukocyte elastase but not of pancreatic elastase. O n the other hand, leukocyte elastase was less effectively inhibited by h u m a n serum than was pancreatic elastase. Subsequently, leukocyte elastase has been purified and other differences have been observed between it and pancreatic elastase.
Leukocyte elastase
appears to exist in multiple forms with different carbohydrate contents, but with identical amino acid compositions (2-5) . The amino acid composition of leukocyte elastase differs from that of pancreatic elastase.
Copyright © 1976 by Academic Press, Inc. All rights o] reproduction in any form reserved.
1327
Preliminary data on the amino-
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
terminal sequences indicate differences between leukocyte elastase and pancreatic elastase (6) . Immunological studies also suggest dlssimilarity(6) . Recently it has been shown that leukocyte elastase appears less active on a molar basis than pancreatic elastase against ligament elastin (6 ,7). The above differences between leukocyte elastase and pancreatic elastase suggest that these enzymes may display qualitative differences in the manner in which they digest elastin. It is possible therefore that features of tissue injury due to leukocyte elastase might not be accurately reflected by studies using pancreatic elastase.
The present study was undertaken to
compare the peptide bond specificities of leukocyte elastase and pancreatic elastase during the catalysis of elastin hydrolysis.
MATERIALS AND METHODS Elastin: Most studies were done with elastin purified from beef neck ligament(8). For studies using porcine pleural elastin, porcine lung parenchymal elastin and porcine tropoelastin the substrates were provided by Dr. L. B. Sandberg (9 'I0). Elastase: Chromatographically pure pancreatic elastase was purchased from Worthington Biochemical Corp., Freehold, N.J. and from Elastin Products, St.Louis, M o . Leukocyte elastase was purified by the method of Baugh and Tr~is from leukocytes collected by leukapheresis from one healthy male age 39'~°1. The enzyme was stored at -70°C after lyophilization. Under these conditions the enzyme retained activity indefinitely. The quantities of enzymes used were determined from extinction coefficients after measuring optical density at 280nM. Fingerprinting of elastin digests: Four milligrams of elastin or tropoelastin were incubated at 37°C with elastase in 0.1 M a m m o n i u m blcarbonate, 0.1 M a m m o n i u m acetate, p H 8.0. The ratios of enzyme to substrate were i:i00 and 1:50 for pancreatic elastase and leukocyte elastase respectively. After two hours an equivalent amount of fresh enzyme was added to the leukocyte elastase reaction mixture. Complete solubilization of substrate occurred after four hours. The reactions were terminated by addition of diisopropylflurophosphate to a final concentration of 5 m M . Two milligrams of solubilized elastin was suspended in 50% pyridine and spotted on W h a t m a n 3 M M paper. Chromatography, followed by high voltage electrophoresis, was performed as described by Sandberg et al (II) . Estimation of the size distribution and the N-terminal amino acids of the peptides in the e l a s t i n d i g e s t s : Fifty milligrams of ligament e l a s t i n was suspended in 1 ml of 0.05 M Tris-HC1 (pH 8.2) and incubated with 100 ug of l e u k o c y t e e l a s t a s e or p a n c r e a t i c e l a s t a s e at 37°C for 24 hours. Eight milligrams of the e l a s t i n d i g e s t s were d i a l y z e d s e p a r a t e l y in 6000-8000 or 12000-14000 molecular weight cutoff membranes (Spectrapor, Spectrum M e d i c a l I n d u s t r i e s , Los Angeles, CA) a g a i n s t 3 c h a n g e s of 50ml of water. The d i a l y s a t e s and the c o n t e n t s of the d i a l y s i s bags
1328
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were evaporated to dryness. Each fraction then underwent hydrolysis and amino acid analysis as previously described(8). To determine the N-terminal amino acids 100ul of elastin digest (5mg) was mixed with 200ul of 0.1 M N a H C O 3 , 100ul dansyl chloride (Img/ml in acetone) and 10ul (10uCi) tritiated dansylchloride (New England Nuclear). The reaction mixture was allowed to dansylate at room temperature for 24 hours. The contents were evaporated employing a flow of nitrogen gas and then hydrolysed in screw top vials using 200ul of 6N HCI at 95°C for 12 hours. The contents were evaporated again and then dissolved in 20ul acetone - glacial acetic acid (4:1 ,v/v). One microliter of the mixture was used for thin layer chromatography on polyamide as described by W o o d s and W a n g (12) . The identity of the dansylated amino acids was established by control chromatograms made with mixtures of pure dansylated amino acids (Sigma Chemical Corp., St. Louis, MO). To quantitate the dansylated amino acids the dansyl spots were cut out, suspended in scintillation fluid and measured for [3H ] activity. RESULTS Chromatograms of digests of elastin and tropoelastin showed differences between the products of pancreatic elastase and leukocyte elastase (Figure i). A prominent component present near the origin in leukocyte elastase digests was not present in the pancreatic elastase digests.
Conversely, the pancreatic
elastase digests had intensely stained zones near the center of the chromatograms which were not seen in the chromatograms of substrates digested by leukocyte elastase. For each enzyme the chromatographic pattern was consistent irrespective of whether the elastin was derived from ligament, lung parenchyma or pleura of bovine or porcine origin. Fingerprints confirmed that leukocyte elastase and pancreatic elastase produce different peptides from elastin and tropoelastin (Figure 2). The prominent zone near the origin on the chromatograms of leukocyte elastase digests was not a single spot after high voltage electrophoresis.
Instead it was dispersed into
many spots along the length of the electrophoretogram which were very faint in elastin digests, but easily seen in tropoelastln digests. In contrast, the heavily stained zone observed near the center of the chromatogram of pancreatic elastase digests appeared to correspond to a single spot after electrophoresis.
1329
Similar
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,
Figure 1 Chromatograms of bovine ligament e l a s t i n d i g e s t e d by leukocyte e l a s t a s e (1) and p a n c r e a t i c e l a s t a s e (2) and t r o p o e l a s t i n digested by l e u k o c y t e e l a s t a s e (3) and p a n c r e a t i c e l a s t a s e (4). The arrow i n d i c a t e s the origin and direction of chromatography. At the top is shown the s e p a r a t i o n of a mixture of amino a c i d s .
to the r e s u l t s with c h r o m a t o g r a p h y , e a c h enzyme produced its d i s t i n c t i v e fingerprint independent of the s o u r c e of the e l a s t i n s u b s t r a t e . D i a l y s i s experiments demonstrated that l e u k o c y t e e l a s t a s e and p a n c r e a t i c e l a s t a s e were similar in digesting a large p e r c e n t a g e of e l a s t i n into peptides with molecular weights below 14000 (Table 1).
The e n z y m e s a l s o resembled one
another in that d e s m o s i n e s were only slightly r e p r e s e n t e d in peptides smaller
1330
Vol. 72, No. 4, 1976
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
+
,+,
,+,y+
+:+,r
+,++
-
+,! + +,
+~,
tI
,
)
2 Figure 2 Fingerprints of bovine ligament elastln digested by pancreatic elastase (I) and leukoctye elastase (2) and tropoelastin digested by pancreatic elastase (3) and leukocyte elastase (4). Chromatography was run in the vertical direction from top to bottom; electrophoresis was run horizontally. Amino acid standards, seen at the bottom of (2) and (4), were included in the electrophoreses to evaluate relative migration.
than 8000 molecular weight, but greater than 50 per cent of the desmoslnes were constituents of peptides in the range 8000-14000 molecular weight. The N-terminal amino acids did not appear to specify the peptide bonds of elastin broken by pancreatic elastase and leukocyte elastase during the hydrolysis of elastin. The N-terminal amino acids and their relative quantities
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TABLE 1 C O M P A R I S O N OF THE DIALYSABLE C O M P O N E N T S OF LIGAMENT ELASTIN AFTER DIGESTION BY PANCREATIC ELASTASE A N D LEUKOCYTE ELASTASE D i a l y s i s (molecular weight range)
6000 -
D i a l y s a b l e weight (per cent of the weight of the t o t a l digest)
Dialysable desmosines (per cent of the d e s m o s i n e s in the t o t a l digest)
Pancreatic Elastase Diqest
Pancreatic Elastase Dige st
Leukocyte Elastase Dige st
Leukocyte Elastase Digest
8000
44
41
Trace
Trace
12000 - 14000
83
76
58
53
TABLE 2 N-TERMINAL A M I N O ACIDS OF ELASTIN DIGESTS* N-Terminal Amino Acids
Elastin Digested By P~ncreatic E l a s t a s e [~H] (cpm)
Elastin Digested By Leukocyte E l a s t a s e [~H] (cpm)
Glycine
31,146
28,775
Alanine
21,250
16,426
Phenylalanine
4,490
4,155
Va line
3,560
4,006
Leucine
2,654
3,061
Proline
1,491
2,093
Isoleucine
1,539
912
*Equal amounts of r a d i o l a b e l e d , d a n s y l a t e d e l a s t i n d i g e s t were s u b j e c t e d to chromatography.
were similar for the peptides of e l a s t i n resulting from digesting by either enzyme (Table 2).
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS DISCUSSION
The p r e s e n t s t u d y d e m o n s t r a t e s t h a t t h e p e p t i d e b o n d s p e c i f i c i t y of leukocyte elastase
on elastin and tropoelastin substrates
pancreatic elastase.
d i f f e r s from t h a t o f
These differences have been demonstrated in chromatograms
a n d f i n g e r p r i n t s of d i g e s t s p r o d u c e d b y t h e t w o e n z y m e s .
That these enzymes
might attack elastin differently is not surprising since other differences between them are known.
It i s of c o n s i d e r a b l e
interest,
however, that mammals should
h a v e t w o a v e n u e s for t h e d i g e s t i o n of s u c h a n u n u s u a l p r o t e i n a s e l a s t i n . The d i s t i n c t i v e f i n g e r p r i n t p a t t e r n s t h a t r e s u l t e d from e l a s t o l y s i s leukocyte elastase the elastin.
and pancreatic elastase
by
w e r e i n d e p e n d e n t of t h e s o u r c e of
E a c h e n z y m e p r o d u c e d a c o n s i s t e n t f i n g e r p r i n t on r e a c t i o n w i t h
e l a s t i n s from s e v e r a l t i s s u e s
from p i g a n d c o w .
These data should not be inter-
p r e t e d t o m e a n t h a t e l a s t i n s a r e i d e n t i c a l in a l l m a m m a l i a n t i s s u e s .
Indeed,
d i f f e r e n c e s in e l a s t i n s a r e k n o w n to e x i s t b e t w e e n s p e c i e s (8) a n d a n t i b o d i e s t o e l a s t i n h a v e b e e n d e v e l o p e d in a n i m a l s (13) . On t h e o t h e r h a n d , t h e f i n g e r p r i n t d a t a s u g g e s t t h a t m a j o r d i f f e r e n c e s i n e l a s t i n s t r u c t u r e from t i s s u e to t i s s u e and between species are unlikely. Although leukocyte elastase is nevertheless
a true elastase.
d i f f e r s from p a n c r e a t i c e l a s t a s e , It d i g e s t e d t h e e l a s t i n
the enzyme
u s e d in t h e s e e x p e r i m e n t s
t o a n e x t e n t s i m i l a r to t h a t a c h i e v e d b y p a n c r e a t i c e l a s t a s e
and it h a s b e e n
s h o w n c a p a b l e o f d i g e s t i n g a v a r i e t y of e l a s t i n s l a b e l e d w i t h c h r o m o g e n s or radioisotopes(1,3,7).
Moreover, incubations of leukocyte elastase
with tissues
p r o d u c e h i s t o l o g i c e v i d e n c e o f t h e b r e a k d o w n of e l a s t i c f i b e r s (14) . L e u k o c y t e elastase
is inhibited by active site directed chloromethylketones which
s p e c i f i c a l l y i n h i b i t p a n c r e a t i c e l a s t a s e (15) . Pancreatic elastase of elastolysis
h a s b e e n u s e d a s a m o d e l of t h e p o s s i b l e e f f e c t s
upon tissues,
n o t a b l y in e x p e r i m e n t a l e m p h y s e m a ( 1 6 ) . It s e e m s
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
much more l i k e l y , however, that l e u k o c y t e e l a s t a s e , rather than p a n c r e a t i c e l a s t a s e , may initiate the p a t h o g e n e s i s of d i s e a s e p r o c e s s e s such as pulmonary emphysema and joint damage (17) . The p r e s e n t study i n d i c a t e s that p a n c r e a t i c e l a s t a s e and l e u k o c y t e e l a s t a s e have different e f f e c t s a g a i n s t e l a s t i n .
Thus,
c a u t i o n should be e x e r c i s e d in the extrapolation of experimental r e s u l t s from p a n c r e a t i c e l a s t a s e to t h o s e r e s u l t s which might o c c u r with l e u k o c y t e e l a s t a s e .
Acknowledgments This work was supported by U S P H S Grant HL 16118. The authors gratefully acknowledge Dr. L. B. Sandberg for elastin and tropoelastin samples and for assistance in fingerprinting.
Reference s i. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17.
Janoff,A. and Scherer, I. (1968) I. Exp. Med. 128:1137-I151. lanoff,A. (1973) Lab. Invest. 2_~9:458-464. Ohlsson, K. and Olsson, I. (1974) Eur. J. Biochem. 4_~2:519-527. Taylor, J.C. and Crawford, I.P. (1975) Arch. Biochem. and Biophys. 169:91-101. Feinstein, G. and lanoff, A. (1975) Biochem. Biophys. Acta 403: 493-505. Baugh, R.J. and Travls, I. (1976) Biochem. i_~5:836-841. Bielefeld,D.R., Senior, R.M. and Yu, S.Y. (1975) Biochem. Biophys. Res. C o m m . 6_/7:1553-1559. Starcher, B.C. and Galione, M.I. (1976) Analytical Biochem., In Press. Rasmussen, B.L., Bruenger, E. and Sandberg, L.B. (1975) Analytical Biochem. 6._44:255-259. Sandberg, L.B., Bruenger, E. and Cleary, E.G. (1975) Analytical Biochem., 6_~4:249-254. Sandberg, L.B., Weissman, N. and Gray, W.R. (1971) Biochem. 1_O0:52-56. W o o d s , K.R. and Wang, K.T. (1967) Biochem. Biophys. Acta 133:369-370. S y k e s , B . C . and Partridge, S . M . (1974) Biochem. J. 14___~1:567-572. Janoff, A. (1970) Lab. I n v e s t . 2__2:228-235. Tuhy, P . M . and Powers, J . C . (1975) FEBS LETTERS 5 0 : 3 5 9 - 3 6 1 . Kaplan, P . D . , Kuhn, C . , and Pierce, J.A. (1973) I. Lab. C l i n . Meal. 82:349-356. lanoff, A . , Blondin, I . , Sandhaus, R . A . , M o s s e r , A . , and Malemud, C. (1975) Proteases and Biological Control, Cold Spring Harbor Conf. Cell Proliferation 2 : 5 0 3 - 6 2 0 .
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