A novel binding site for the native hepatic acute‐phase protein α1‐antitrypsin expressed on the human hepatoma cell line HepG 2 and intestinal cell line Caco 2
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Abstract: Aims/Background: α1‐antitrypsin (α1‐AT) is a hepatic acute phase protein which predominantly inhibits neutrophil elastase. Besides this major function, we have also previously shown that α1‐AT markedly increased H‐ferritin mRNA expression and ferritin synthesis in the human hepatoma cell line HepG 2. These actions suggest that α1‐AT might interact with HepG 2 cells via a specific cell surface binding site. Methods and Results: Using radio‐labelled native α1‐AT, we observed saturable binding to HepG 2 cells with a dissociation constant (Kd) of 63.3±6.9 nM and a maximal density of binding sites (Bmax) of 0.34 ±0.05 pmol/10 6 cells equivalent to 195800±29200 sites/cell. The binding of [ 125 I]α1‐AT was time dependent with a calculated association rate constant of 9.22±1.84×10 4 ×M −1 ×min −1 . Binding was highly specific since other acute phase proteins or protease inhibitors failed to block binding. Although α1‐AT‐trypsin, α1‐AT‐elastase and the pentapeptide FVYLI, the minimal binding sequence for the SEC receptor, increased [ 125 I]α1‐AT binding, in long term experiments these complexes failed to influence the number of α1‐AT binding sites. Specific, saturable binding of [ 125 I]α1‐AT was also found on the human intestinal epithelial Caco 2 cells, but not on fibroblast or leukaemic cell lines. Conclusion: These experiments demonstrate a specific, high affinity binding site for native α1‐AT on HepG 2 and Caco 2 cells, cell lines derived from tissues involved in the acute phase response.Keywords:
Pancreatic elastase
Dissociation constant
Airway instillation of proteinases with the ability to degrade elastin has been used to produce disease in the rat analogous to human pulmonary emphysema. This study examined the retention, localization, and fate of endotracheally instilled elastase using 125Iodine labeled enzyme and immunoperoxidase histochemistry. Porcine pancreatic elastase labeled with 125I was detected in rat lungs through 96 h after instillation; over half of the label was still present after 7 h. Similar results were obtained when elastase was reacted with a specific, catalytic site inactivator prior to instillation. Trypsin and denatured elastase, however, were cleared much more rapidly from the lung (less than half of the label present after 30 min). When lungs were homogenized after instillation of active elastase, the soluble fraction contained elastase bound to rat alpha1-antitrypsin. In addition, a small amount of label (less than 10%) appeared bound to insoluble components for extended periods of time. Using immunoperoxidase histochemistry, it was found that exogenous elastase was rapidly contained with pulmonary alveolar macrophages, as well as associated with alveolar septums and other parenchymal structures. Similar results were obtained with elastase from both porcine pancreas and human neutrophils. These results suggest that exogenous elastase in the rat, and perhaps endogenous elastolytic enzymes in humans, may have several fates in the lungs: complex formation with endogenous inhibitors, containment within the macrophage, and/or association with connective tissue targets.
Pancreatic elastase
Immunoperoxidase
Alveolar macrophage
Pulmonary alveolus
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A radioimmunoassay for porcine, cationic, pancreatic elastase (irPE) is described. Normal porcine serum contains only small amounts of irPE (<3 µg/l). IrPE in serum and peritoneal exudate from 6 pigs with experimental pancreatitis was found mainly in a molecular form corresponding to free pro-enzyme. The presence of α1-, α2-macroglobulin-bound elastase-like enzymatic activity in the peritoneal exudates from pigs with pancreatitis, however, indicates that the proteinase is to some extent released as the active enzyme. In some pigs with pancreatitis, the elastase-like activity against Succ(Ala) in the peritoneal exudates increased during the experiment, arguing for a progressive activation of pro-elastase. Free proteolytic activity was not observed in any of the peritoneal exudates. This low degree of activation of elastase and the fact that the elastase inhibiting capacity is substantially larger than the trypsin inhibiting capacity in serum and biological fluids, leads us to the conclusion that active elastase is not a factor of principal importance in the pathogenesis of proteinase inhibitor consumption and tissue damage in our experimental pancreatitis model.
Pancreatic elastase
Pathogenesis
Peritoneal fluid
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Pancreatic elastase
Digestion
Proteolysis
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1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.
alpha-2-Macroglobulin
Pancreatic elastase
Alpha (finance)
Protease inhibitor (pharmacology)
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Incubation of human serum α 1 ‐antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. α 1 ‐Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor:enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when α 1 ‐antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex ( M r near 77000) and the release of a fragment migrating as a peptide of M r near 5000. Moreover a free proteolytically modified form of α 1 ‐antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when α 1 ‐antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of α 1 ‐antichymotrypsin, so far limited to the inhibition of chymotrypsin‐like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of α 1 ‐antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.
Pancreatic elastase
Cathepsin G
Alpha (finance)
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Pancreatic elastase
Proteinase inhibitor
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Varying amounts of crystalline porcine pancreatic elastase were injected iv into puppies in order to study its effect in vivo on elastic tissue (21) and on serum elastase inhibitors. Serum inhibitors to pancreatic elastase and to trypsin, were determined in blood samples drawn at intervals after injection. The serum elastase inhibition level decreased 3-5 hr after elastase injection. The decrease was a function of the amount of enzyme injected and reached the highest levels at 80-200 mg elastase injected. The decrease of elastase inhibitor titer with injected elastase suggests a dissociable enzyme-inhibitor complex (es). No parallelism was observed between serum inhibitor titers to elastase and to trypsin. The trypsin inhibitor titer dropped only exceptionally and did not follow at any time the kinetics of the elastase inhibitors. The elastase inhibitor titer returned to normal 24 hr after injection of 20-150 mg of enzyme but only partial recovery was observed in this interval with higher doses. The relatively low affinity of the serum inhibitors to elastase may explain the tissue lesions observed even at low doses of elastase injected (20 mg).
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Elastase was purified from an acetone-ether powder of porcine pancreas by a one step affinity chromatography procedure on IgG-Sepharose 4B. The IgG was derived from a rabbit immunized with porcine pancreatic elastase and was itself isolated by affinity chromatography on elastase immobilized on the same matrix. The column was calibrated with a known elastase preparation under standardized conditions. Direct isolation of elastase from pancreatic extracts yielded approximately 90 mg from two pancreas. The enzymatic activity and electrophoretic migration in SDS-polyacrylamide gel of the purified elastase were equal to those of the purest commercially available enzymes. Application of this method to aorta of young pigs suggests the presence of active elastase in the aorta.
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Sepharose
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Pancreatic elastase
Enzyme Kinetics
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Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.
Pancreatic elastase
alpha-2-Macroglobulin
Alpha (finance)
Protease inhibitor (pharmacology)
Neutrophil elastase
Mesocricetus
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