Biol. Chem. Hoppe-Seyler Vol. 371, pp. 441-446, May 1990
Susceptibility of Baboon Aorta Elastin to Proteolysis Lucienne DESFONTAINES, William HORNEBECK, Sao Ming WEI, Ladislas ROBERT and Chantal LAFUMA Laboratoire de Biochimie duTissu Conjonctif, UACNRS 1174, Faculte de Medecine, Creteil
(Received 10 January 1990)
Summary: Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants Fand [S50] of aortic elastin hydrolysis by HLE (0.15 ) were 0.00286 1 mg x x min"1 and 0.158 mg x m/'1, respectively. One mg of this elastin could be saturated with 5.6 ^g of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was
highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine -chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.
Empfindlichkeit von Pavianaorten-E lastin gegenüber Proteinasen Zusammenfassung: Elastin wurde mit Hilfe von Achromobacter-Kollagenase aus Pavianaorten gereinigt und seine Empfindlichkeit gegenüber verschiedenen Proteinasen untersucht. Elastase aus menschlichen Leukozyten (HLE) hydrolysierte das Elastin 8mal schneller als menschliches Cathepsin G; RinderChymotrypsin war völlig unwirksam. Die kinetischen Konstanten der Elastin-Hydrolyse mit HLE (0.15 ) waren V= 0.00286 mg x m/'1 x min'1 und [S50] = 0.158 mg x m/"1. 5.6 ^g HLE sättigten l mg Elastin. Ähnlich wie bei aus anderen Organen isolierten Elastinen ist die Hydrolysegeschwindigkeit des
Pavianaorten-Elastins von der lonenstärke stark abhängig, wobei dieser Effekt biphasisch ist. Die Elastolyse ist bei 0.15M NaCl fast auf das Doppelte beschleunigt. Weitere Erhöhung der lonenstärke führt zu einer Verminderung der Adsorption der Elastase an das Substrat und zu einer Abnahme der Geschwindigkeit der Elastolyse. Cathepsin G, nicht aber Rinder-a-Chymotrypsin, stimuliert die Hydrolyse des Elastins durch HLE. Im molekularen Verhältnis l: l angewandt, stimuliert Cathepsin G l.Tfach, und im Verhältnis 8:1 2.1fach die Wirkung der HLE.
Enzymes: Bovine chymotrypsin (EC 3.4.21.1); Human leukocyte elastase (EC 3.4.21.37); Porcine pancreatic elastase (EC 3.4.21.36); Human leukocyte cathepsin G (EC 3.4.21.20). Abbreviations: Cat. G., Human leukocyte cathepsin G; Des, desmosine; HLE, human leukocyte elastase; Ides, isodesmosine; MeO-Suc, 4-methoxycarbonyl propionyl; Nan, 4-nitroanilide; PPE, porcine pancreatic elastase.
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442
L. Desfontaines,W. Hornebeck, S.M.Wei, L. Robert andC. Lafuma
Vol. 371 (1990)
Key words: Elastases, elastin, aorta, proteolysis.
Arteriosclerosis is characterized by an intense fragmentation of elastic lamellae which has been attributed to the hydrolysis of aortic media elastin by elastolytic proteinases synthesized by several cell types11·21. Only one elastin gene has been identified, but the diversity of elastin structures in tissues and/or species could arise from alternative splicing of a primary transcript'31.This, together with variations in the extent of the post translational modifications of elastin such as desmosine formation and hydroxyproline content could lead to the formation of elastic fibers with different susceptibilities to proteolysis141. The varying susceptibilities to elastolysis among elastins of different origins could be masked by the harsh procedures generally used to purify this matrix macromolecule e.g. the hot alkali treatment, or autoclaving[5]. We therefore isolated elastin from the media of baboon aorta using a mild extraction procedure and collagen digestion by Achromobacter collagenase161 followed by only a short treatment with hot alkali. This report describes the susceptibility to proteolysis of this aortic elastin; the kinetic parameters, the salt effect, and stimulation by cathepsin G of baboon aortic elastin degradation by human leukocyte elastase were compared to similar parameters obtained with elastins from other sources. Materials and Methods Chemicals The enzyme substrate Suc-[Ala]2-Pro-Phe-Nan, thermolysin (batch no. P-1512, spec, activity 59 U/mg) and bovine chymotrypsin were purchased from Sigma Chemicals (St. Louis, MO 63178, U.S.A.). Suc-[Ala] r Nan and porcine pancreatic elastase (Batch no. 8010, spec, activity 17.4 U/mg) were obtained from CHOAY Laboratories (F-75015 Paris). Human leukocyte elastase was from Elastin Products Co. (St. Louis, MO 63100, U.S.A.) and [ 3 H]NaBH 4 (Sp. act. 20 Ci/mmol was from CEA (F-91191 Saclay). Human cathepsin G was a generous gift of C. Boadier and/. Bieth (Faculte de Pharmacie, F-67048 Strasbourg) and eglin C was provided by H.R Schnebli, (Ciba Geigy, CH-4002 Basel). The purification of Achromobacter collagenase was performed in the laboratory of Professor Keil^ (Institut Pasteur, F-75724 Paris). All other chemicals of highest purity available were from Merck (D6100 Darmstadt). Purification and radiolabeling of baboon aorta elastin Baboon aortae were studied because of their relative similarity to human aortae and the use of these species in atherosclerosis research. Thoracic and abdominal aortas were obtained from 8 baboons (Papio papio) from West Africa, weighing 4.5 ± 0.5 kg. The adventitia of baboon aortae was stripped off and the media of every aorta (5.4 g wet weight) was homogenized in 0.15M NaCl at 4 °C. Salt soluble proteins were extracted 3 times for 24 h.The residue was delipidated with chloroform/methanol 2:1 (v/v) at 4 °C during
6 h and further submitted to 3 successive extractions with 5\1 guanidinium chloride, 0.05M dithiothreitol in lOOmM Tris/HCl pH 8.0. The insoluble material (3.2 g) was hydrolysed with Achromobactercollagenase (1:1000 (w/w)hi lOOmMTris, lOmw CaCl2 pH 8.5 during 27 h at 37 °C. These conditions have been shown to completely remove collagen from baboon aorta and lung. The residue still contained elastin-associated microfibrils which were eliminated by 3 extractions at 4 °C with 5M guanidinium chloride, 0.05M dithiothreitol in lOOmMTris/HCl pH 8.0, followed by one treatment with 0.1 M NaOH at 98 °C for 15 min. The residue (0.42 g) was exhaustively washed with distilled water, ethanol and dried with acetone. Insoluble elastin was then finely crushed with a Potter and calibrated to 30-60 mesh. This purification schedule gave reproducible results (relative amount of elastin and amino-acid composition). All buffers, with the exception of those used for collagenase digestion, contained proteinase inhibitors e.g. phenylmethanesulfonylfluoride, N-ethylmaleimide and ethylenediamine tetraacetic acid (ImMeach). The amino-acid composition of the aortic elastin preparation was evaluated by the Pico-Tag method; the phenyl-thiocarbamoylamino acids were separated on a C18 column using a 5-60% acetonitrile gradient' 7 '. A special program was devised in order to separate and quantify desmosine crosslinks'8'. Aortic elastin was radiolabelled with [ 3 H]NaBH 4 as previously described by Rifkin et al.'9'. Its specific activity was 370 kBq/mg. Proteinase assays Human leukocyte elastase, bovine chymotrypsin and cathepsin G were titrated with eglin c which reacts stoichiometrically (1:1) with these proteinases' '"'.Themolarity of porcine pancreatic elastase was determined with trypsin-titrated
Susceptibility of Baboon Aorta Elastin to Proteolysis Lucienne DESFONTAINES, William HORNEBECK, Sao Ming WEI, Ladislas ROBERT and Chantal LAFUMA Laboratoire de Biochimie duTissu Conjonctif, UACNRS 1174, Faculte de Medecine, Creteil
(Received 10 January 1990)
Summary: Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants Fand [S50] of aortic elastin hydrolysis by HLE (0.15 ) were 0.00286 1 mg x x min"1 and 0.158 mg x m/'1, respectively. One mg of this elastin could be saturated with 5.6 ^g of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was
highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine -chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.
Empfindlichkeit von Pavianaorten-E lastin gegenüber Proteinasen Zusammenfassung: Elastin wurde mit Hilfe von Achromobacter-Kollagenase aus Pavianaorten gereinigt und seine Empfindlichkeit gegenüber verschiedenen Proteinasen untersucht. Elastase aus menschlichen Leukozyten (HLE) hydrolysierte das Elastin 8mal schneller als menschliches Cathepsin G; RinderChymotrypsin war völlig unwirksam. Die kinetischen Konstanten der Elastin-Hydrolyse mit HLE (0.15 ) waren V= 0.00286 mg x m/'1 x min'1 und [S50] = 0.158 mg x m/"1. 5.6 ^g HLE sättigten l mg Elastin. Ähnlich wie bei aus anderen Organen isolierten Elastinen ist die Hydrolysegeschwindigkeit des
Pavianaorten-Elastins von der lonenstärke stark abhängig, wobei dieser Effekt biphasisch ist. Die Elastolyse ist bei 0.15M NaCl fast auf das Doppelte beschleunigt. Weitere Erhöhung der lonenstärke führt zu einer Verminderung der Adsorption der Elastase an das Substrat und zu einer Abnahme der Geschwindigkeit der Elastolyse. Cathepsin G, nicht aber Rinder-a-Chymotrypsin, stimuliert die Hydrolyse des Elastins durch HLE. Im molekularen Verhältnis l: l angewandt, stimuliert Cathepsin G l.Tfach, und im Verhältnis 8:1 2.1fach die Wirkung der HLE.
Enzymes: Bovine chymotrypsin (EC 3.4.21.1); Human leukocyte elastase (EC 3.4.21.37); Porcine pancreatic elastase (EC 3.4.21.36); Human leukocyte cathepsin G (EC 3.4.21.20). Abbreviations: Cat. G., Human leukocyte cathepsin G; Des, desmosine; HLE, human leukocyte elastase; Ides, isodesmosine; MeO-Suc, 4-methoxycarbonyl propionyl; Nan, 4-nitroanilide; PPE, porcine pancreatic elastase.
Brought to you by | Purdue University Libraries Copyright © by WalterAuthenticated de Gruyter & Co · Berlin · New York
Download Date | 5/20/15 10:39 AM
442
L. Desfontaines,W. Hornebeck, S.M.Wei, L. Robert andC. Lafuma
Vol. 371 (1990)
Key words: Elastases, elastin, aorta, proteolysis.
Arteriosclerosis is characterized by an intense fragmentation of elastic lamellae which has been attributed to the hydrolysis of aortic media elastin by elastolytic proteinases synthesized by several cell types11·21. Only one elastin gene has been identified, but the diversity of elastin structures in tissues and/or species could arise from alternative splicing of a primary transcript'31.This, together with variations in the extent of the post translational modifications of elastin such as desmosine formation and hydroxyproline content could lead to the formation of elastic fibers with different susceptibilities to proteolysis141. The varying susceptibilities to elastolysis among elastins of different origins could be masked by the harsh procedures generally used to purify this matrix macromolecule e.g. the hot alkali treatment, or autoclaving[5]. We therefore isolated elastin from the media of baboon aorta using a mild extraction procedure and collagen digestion by Achromobacter collagenase161 followed by only a short treatment with hot alkali. This report describes the susceptibility to proteolysis of this aortic elastin; the kinetic parameters, the salt effect, and stimulation by cathepsin G of baboon aortic elastin degradation by human leukocyte elastase were compared to similar parameters obtained with elastins from other sources. Materials and Methods Chemicals The enzyme substrate Suc-[Ala]2-Pro-Phe-Nan, thermolysin (batch no. P-1512, spec, activity 59 U/mg) and bovine chymotrypsin were purchased from Sigma Chemicals (St. Louis, MO 63178, U.S.A.). Suc-[Ala] r Nan and porcine pancreatic elastase (Batch no. 8010, spec, activity 17.4 U/mg) were obtained from CHOAY Laboratories (F-75015 Paris). Human leukocyte elastase was from Elastin Products Co. (St. Louis, MO 63100, U.S.A.) and [ 3 H]NaBH 4 (Sp. act. 20 Ci/mmol was from CEA (F-91191 Saclay). Human cathepsin G was a generous gift of C. Boadier and/. Bieth (Faculte de Pharmacie, F-67048 Strasbourg) and eglin C was provided by H.R Schnebli, (Ciba Geigy, CH-4002 Basel). The purification of Achromobacter collagenase was performed in the laboratory of Professor Keil^ (Institut Pasteur, F-75724 Paris). All other chemicals of highest purity available were from Merck (D6100 Darmstadt). Purification and radiolabeling of baboon aorta elastin Baboon aortae were studied because of their relative similarity to human aortae and the use of these species in atherosclerosis research. Thoracic and abdominal aortas were obtained from 8 baboons (Papio papio) from West Africa, weighing 4.5 ± 0.5 kg. The adventitia of baboon aortae was stripped off and the media of every aorta (5.4 g wet weight) was homogenized in 0.15M NaCl at 4 °C. Salt soluble proteins were extracted 3 times for 24 h.The residue was delipidated with chloroform/methanol 2:1 (v/v) at 4 °C during
6 h and further submitted to 3 successive extractions with 5\1 guanidinium chloride, 0.05M dithiothreitol in lOOmM Tris/HCl pH 8.0. The insoluble material (3.2 g) was hydrolysed with Achromobactercollagenase (1:1000 (w/w)hi lOOmMTris, lOmw CaCl2 pH 8.5 during 27 h at 37 °C. These conditions have been shown to completely remove collagen from baboon aorta and lung. The residue still contained elastin-associated microfibrils which were eliminated by 3 extractions at 4 °C with 5M guanidinium chloride, 0.05M dithiothreitol in lOOmMTris/HCl pH 8.0, followed by one treatment with 0.1 M NaOH at 98 °C for 15 min. The residue (0.42 g) was exhaustively washed with distilled water, ethanol and dried with acetone. Insoluble elastin was then finely crushed with a Potter and calibrated to 30-60 mesh. This purification schedule gave reproducible results (relative amount of elastin and amino-acid composition). All buffers, with the exception of those used for collagenase digestion, contained proteinase inhibitors e.g. phenylmethanesulfonylfluoride, N-ethylmaleimide and ethylenediamine tetraacetic acid (ImMeach). The amino-acid composition of the aortic elastin preparation was evaluated by the Pico-Tag method; the phenyl-thiocarbamoylamino acids were separated on a C18 column using a 5-60% acetonitrile gradient' 7 '. A special program was devised in order to separate and quantify desmosine crosslinks'8'. Aortic elastin was radiolabelled with [ 3 H]NaBH 4 as previously described by Rifkin et al.'9'. Its specific activity was 370 kBq/mg. Proteinase assays Human leukocyte elastase, bovine chymotrypsin and cathepsin G were titrated with eglin c which reacts stoichiometrically (1:1) with these proteinases' '"'.Themolarity of porcine pancreatic elastase was determined with trypsin-titrated
