scholarly journals Modification of the tandem reactive centres of human inter-α-trypsin inhibitor with butanedione and cis-dichlorodiammineplatinum(II)

1988 ◽  
Vol 254 (1) ◽  
pp. 171-178 ◽  
Author(s):  
M W Swaim ◽  
S V Pizzo
Keyword(s):  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Proteinase Inhibitor ◽  
Complete Loss ◽  
Cathepsin G ◽  
Partial Loss ◽  
Inhibitory Specificity ◽  
Region Sequence ◽  
Leucocyte Elastase ◽  
Kunitz Type

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.

scholarly journals Purification of a serine-proteinase inhibitor from human articular cartilage. Identity with the acid-stable proteinase inhibitor of mucous secretions

1991 ◽  
Vol 274 (1) ◽  
pp. 269-273 ◽  
Author(s):  
B Böhm ◽  
R Deutzmann ◽  
H Burkhardt
Keyword(s):  
Articular Cartilage ◽  
Proteinase Inhibitor ◽  
Serine Proteinase ◽  
Cathepsin G ◽  
Human Articular Cartilage ◽  
Serine Proteinase Inhibitor ◽  
Inhibitor Molecule ◽  
Leucocyte Elastase ◽  
Sds Page

An inhibitor of the serine proteinases human leucocyte elastase (EC 3.4.21.37), of cathepsin G (EC 3.4.21.20) and of trypsin (EC 3.4.21.4) has been purified from human articular cartilage. The apparent Mr of the cationic (pI greater than 10) protein was determined to 15,000 by SDS/PAGE. It was shown to cross-react in Western blot with a specific antibody to a recombinant-derived serine-proteinase inhibitor of human mucous secretions. Identity of both inhibitors is indicated by the determination of the N-terminal amino acid sequence of the cartilage-derived serine-proteinase inhibitor. In all 24 residues the cartilage inhibitor was shown to be identical with the human secretory leucocyte proteinase inhibitor (‘SLPI’). The inhibitor molecule may play a crucial role in the protection of cartilage matrix proteins against proteolytic attack.

scholarly journals A Retrospective Analysis of the Cartilage and Intervertebral Disc Kunitz Protease Inhibitory Proteins Identifies These as Members of The Inter-α-Trypsin Inhibitor Superfamily with potential roles in the protection of the articulatory surface

2018 ◽  
Author(s):  
Susan Smith ◽  
James Melrose
Keyword(s):  
Articular Cartilage ◽  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Concanavalin A ◽  
Inhibitory Activity ◽  
Proteinase Inhibitors ◽  
Cathepsin G ◽  
Prolonged Storage ◽  
Lectin Affinity Chromatography ◽  
Lectin Affinity

The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Ovine articular cartilage was finely diced and extracted in 6M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity and pooled fractions assessed by affinity blotting using biotinylated trypsin to detect active SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS stub epitope generated by chondroitinase-ABC digestion. This identified 2-B-6 (+) positive 220-250,120, 58 and 36 kDa SPIs. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulphate stub epitope consistent with its identity as the α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa could be autolytically generated by prolonged storage of the aforementioned 120 and 58 kDa SPIs; chymotrypsin affinity chromatography also generated the 6kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 both displayed potent inhibitory activity towards trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and HA in the surface regions of articular cartilage represented probable binding sites for the ITI SPs with likely importance in the preservation of joint function.

scholarly journals Human bronchial leucocyte proteinase inhibitor. Rapid isolation and kinetic analysis with human leucocyte proteinases

1985 ◽  
Vol 225 (2) ◽  
pp. 463-472 ◽  
Author(s):  
C E Smith ◽  
D A Johnson
Keyword(s):  
Proteinase Inhibitor ◽  
Dissociation Constants ◽  
Gel Filtration ◽  
Single Step ◽  
Cathepsin G ◽  
Polymorphonuclear Leucocytes ◽  
Bovine Trypsin ◽  
Leucocyte Elastase ◽  
Equilibrium Dissociation Constants

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 × 10(-10) M, 4.18 × 10(-9) M, 8.28 × 10(-9) M and 2.63 × 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.

Activation of latent collagenase from polymorphonuclear leukocytes by cathepsin G

1979 ◽  
Vol 44 (10) ◽  
pp. 3177-3182 ◽  
Author(s):  
Mária Stančíková ◽  
Karel Trnavský
Keyword(s):  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Polymorphonuclear Leukocytes ◽  
Soya Bean ◽  
Cathepsin G ◽  
Sepharose Column ◽  
Bean Trypsin Inhibitor ◽  
Human Polymorphonuclear Leukocytes

Cathepsin G was isolated from human polymorphonuclear leukocytes and purified by affinity chromatography on Antilysin-Sepharose column. Purified enzyme activated later collagenase isolated from leukocytes. Activation at 36°C was maximal after 30 min incubation. Inhibitors of cathepsin G - soya-bean trypsin inhibitor, diisopropyl phosphofluoridate and Antilysin were active in inhibiting the activation of latent collagenase by cathepsin G.

scholarly journals Identification and Target-Modification of SL-BBI: A Novel Bowman–Birk Type Trypsin Inhibitor from Sylvirana latouchii

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1254 ◽  
Author(s):  
Xi Chen ◽  
Dong Chen ◽  
Linyuan Huang ◽  
Xiaoling Chen ◽  
Mei Zhou ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Membrane Damage ◽  
Antiproliferative Effects ◽  
Docking Simulations ◽  
Cell Membrane Damage ◽  
Action Mode ◽  
Trypsin Inhibition ◽  
Cloning Method

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

scholarly journals Active fragments obtained by cyanogen bromide cleavage of ovomucoid

1976 ◽  
Vol 155 (2) ◽  
pp. 345-351 ◽  
Author(s):  
J G. Beeley
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Cyanogen Bromide ◽  
Peptide Chain ◽  
Terminal Portion ◽  
Trypsin Inhibitory Activity ◽  
Methionine Residues ◽  
Active Fragments

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.

Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds

1996 ◽  
Vol 41 (4) ◽  
pp. 1017-1022 ◽  
Author(s):  
Isabel F.C Batista ◽  
Maria Luiza V Oliva ◽  
Mariana S Araujo ◽  
Misako U Sampaio ◽  
Michael Richardson ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Primary Structure ◽  
Kunitz Type
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