Inactivation of human alpha 1-proteinase inhibitor by thiol proteinases.
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ABSTRACT:
SUBMITTER: Johnson D
PROVIDER: S-EPMC1164746 | biostudies-other | 1977 Jun
REPOSITORIES: biostudies-other
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Project description:1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.
| S-EPMC1163859 | biostudies-other
Project description:Bronchial leucocyte proteinase inhibitor (BLPI) is an 11 000 Mr protein found in human mucous secretions. This inhibitor apparently controls the serine proteinases elastase and cathepsin G, released from extravascular polymorphonuclear leucocytes. A simple, single-step chromatographic procedure for the isolation of BLPI based on its affinity for chymotrypsin was developed. The purified inhibitor was homogeneous by electrophoresis and gel filtration. Amino acid analyses were in close agreement with previous reports, and showed BLPI to be rich in proline and cystine, but lacking histidine. We have further characterized the role of BLPI with respect to human leucocyte elastase and cathepsin G by close examination of the kinetic parameters. Additionally, we have determined the kinetics of association (kon) and dissociation (koff) for BLPI with bovine trypsin and chymotrypsin. Equilibrium dissociation constants (Ki) of 1.87 X 10(-10) M, 4.18 X 10(-9) M, 8.28 X 10(-9) M and 2.63 X 10(-8) M were obtained for human leucocyte elastase, cathepsin G, bovine trypsin and chymotrypsin, respectively. These results are discussed with respect to BLPI's possible function in vivo and its role relative to other inhibitors in bronchial secretions.
| S-EPMC1144612 | biostudies-other
Project description:Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.
| S-EPMC1183670 | biostudies-other
Project description:Both the clotting and esterase activities of thrombin are inhibited by alpha1-proteinase inhibitor (alpha1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of alpha1-proteinase inhibitor are involved. alpha1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as alpha1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.
| S-EPMC1164146 | biostudies-other
Project description:The kinetic investigation of the inhibition of human pancreatic trypsin 1, trypsin 2 and chymotrypsin A by mucus proteinase inhibitor, eglin c and aprotinin reveals that (i) the first protein is a potent inhibitor of chymotrypsin A (kass. = 1.4 x 10(6) M-1.s-1, Ki = 71 pM) but forms loose complexes with trypsin 1 (Ki = 0.5 microM) and trypsin 2 (Ki = 18 nM), (ii) eglin c does not inhibit the two trypsins but forms a tight complex with chymotrypsin A (kass. = 3.3 x 10(6) M-1.s-1, Ki < 0.1 nM) and (iii) aprotinin is a potent inhibitor of trypsin 1 (kass. = 1 x 10(6) M-1.s-1, Ki < 0.2 nM) and trypsin 2 (kass. = 2.4 x 10(5) M-1.s-1, Ki < 1 nM) but forms a loose complex with chymotrypsin A (Ki = 0.17 microM). These data, together with those published previously on human pancreatic elastase, suggest that a cocktail of aprotinin + eglin c might be a better intensive-care drug for acute pancreatitis than aprotinin alone, because it will efficiently inhibit all four human pancreatic proteinases. On the other hand, human gastric juice inactivates mucus proteinase inhibitor by pepsin-mediated cleavage. This indicates that the fraction of mucus proteinase inhibitor that reaches the stomach following aerosol delivery to cystic fibrosis patients does not reach the duodenum in an active form and, therefore, does not aggravate the pancreatic insufficiency of these patients.
| S-EPMC1216943 | biostudies-other
Project description:INTRODUCTION: Recombinant alpha(1)-proteinase inhibitor, clinically developed for inhalative augmentation therapy in patients with alpha(1)-proteinase inhibitor deficiency or cystic fibrosis, may directly contribute to leukocyte accumulation as it may function as a chemoattractant. The migratory effects of yeast-derived human recombinant alpha(1)-proteinase inhibitor on human peripheral blood neutrophils and eosinophils were therefore tested in vitro. MATERIALS AND METHODS: Human peripheral blood leukocytes were prepared from forearm venous blood and tested for migration toward various preparations of yeast-derived recombinant alpha(1)-proteinase inhibitor in modified Boyden-chamber micropore filter assays. RESULTS: No direct effects of yeast-derived recombinant human alpha(1)-proteinase inhibitor on in vitro migration of isolated neutrophils or eosinophils were seen. CONCLUSIONS: The lack of direct chemotactic effects of recombinant human alpha(1)-proteinase inhibitor despite anti-inflammatory effects in other biological activities of leukocytes may contribute to the preserved antibacterial defense mechanisms observed in patients under experimental augmentation therapy with inhaled alpha(1)-proteinase inhibitor.
| S-EPMC2766450 | biostudies-other
Project description:The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.
| S-EPMC1153604 | biostudies-other
Project description:Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.
| S-EPMC1148236 | biostudies-other
Project description:BackgroundHuman alpha1-proteinase inhibitor (alpha1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alpha1-PI (r-alpha1-PI) could provide an attractive alternative. Although r-alpha1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.ResultsWe have explored the possibility of expressing the gene for human alpha1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha1-PI was similar to that of the pd-alpha1-PI. In vitro stability of the r-alpha1-PI from A. niger was tested in comparison with pd-alpha1-PI reference and non-glycosylated human r-alpha1-PI from E. coli.ConclusionWe examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.
| S-EPMC2186354 | biostudies-literature
Project description:Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.
| S-EPMC2974786 | biostudies-literature