Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor

2010 ◽  
Vol 391 (5) ◽  
pp. 561-570 ◽  
Author(s):  
Larissa P. Coppini ◽  
Nilana M.T. Barros ◽  
Marcela Oliveira ◽  
Izaura Y. Hirata ◽  
Marcio F.M. Alves ◽  
...  
Keyword(s):  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Resonance Energy ◽  
Peptide Bond ◽  
Fibrin Clot ◽  
Cathepsin S ◽  
Enzyme Specificity ◽  
Amino Terminal ◽  
Cathepsin V

Abstract Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S3′-P3′ interaction indicates the significance of the extended subsites for enzyme specificity and affinity.

Radioimmunoassay for the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bone collagen degradation

1993 ◽  
Vol 39 (4) ◽  
pp. 635-640 ◽  
Author(s):  
J Risteli ◽  
I Elomaa ◽  
S Niemi ◽  
A Novamo ◽  
L Risteli
Keyword(s):  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bone Collagen ◽  
Collagen Molecule ◽  
Type I ◽  
Serum Samples ◽  
Amino Terminal ◽  
Wide Range ◽  
Carboxy Terminal

Abstract We developed a radioimmunoassay (RIA) for the carboxy-terminal telopeptides of type I collagen (ICTP), cross-linked with the helical domain of another type I collagen molecule, after isolation from human femoral bone. The cross-linked peptide was liberated by digesting insoluble, denatured bone collagen either with bacterial collagenase or with trypsin, and purified by two successive reversed-phase separations on HPLC, with monitoring of pyridinoline-specific fluorescence. The purity of the peptide was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its origin in the type I collagen fibers was determined by amino-terminal amino acid sequencing. Polyclonal antibodies and a separation reagent containing second antibody and polyethylene glycol are used in the RIA. An immunologically identical, somewhat larger antigen is present in human serum; its concentration increases in multiple myeloma and in rheumatoid arthritis. The ICTP antigen seems to be cleared from the circulation by the kidneys, because glomerular filtration rates that are two-thirds of normal or less are associated with increased circulating ICTP concentrations. The CVs of the method are between 3% and 8% for a wide range of concentrations. The analysis of 40 serum samples can be completed in 4 h.

scholarly journals A Novel Degradation Pathway of Tissue Factor Pathway Inhibitor: Incorporation Into Fibrin Clot and Degradation by Thrombin

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1883-1892 ◽  
Author(s):  
Naoki Ohkura ◽  
Kei-ichi Enjyoji ◽  
Yu-ichi Kamikubo ◽  
Hisao Kato
Keyword(s):  
Tissue Factor ◽  
Calcium Ion ◽  
Tissue Factor Pathway Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fibrin Clot ◽  
Degradation Pathway ◽  
Normal Plasma ◽  
Tissue Factor Pathway ◽  
Basic Region

Abstract Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF ) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from antithrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).

Clot-embedded natural surfactant: kinetics of fibrinolysis and surface activity

1994 ◽  
Vol 267 (5) ◽  
pp. L618-L624 ◽  
Author(s):  
A. Gunther ◽  
M. Kalinowski ◽  
A. Elssner ◽  
W. Seeger
Keyword(s):  
Surface Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lung Surfactant ◽  
Sodium Dodecyl ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Natural Surfactant ◽  
Clot Material ◽  
Kinetics Of

Polymerization of fibrin in the presence of pulmonary surfactant was recently noted to induce incorporation of phospholipids into the insoluble clot material, thereby effecting severe loss of surface activity (W. Seeger, A. Elssner, A. Gunther, H.-J. Kramer, and H. O. Kalinowski. Am. J. Respir. Cell Mol. Biol. 9: 213'220, 1993). In the present study, we investigated the influence of such incorporation of calf lung surfactant extract (CLSE) on the enzymatic cleavage of the fibrin network with the use of plasmin, trypsin, or elastase. Employing a fibrin-plate assay, the proteolytic release of radioactivity originating from 125I-labeled fibrinogen was assessed, and the pattern of split products was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. Surface activity of CLSE was measured in the pulsating bubble surfactometer. When incorporated into the fibrin clot, CLSE inhibited the cleavage of fibrin by all proteases in a dose-dependent manner without affecting the profile of scission products. Inhibition of plasmin-induced clot lysis was also noted on incorporation of CLSE into clotted plasma and on incorporation of dipalmitoylphosphatidylcholine into fibrin polymers. In contrast, corresponding concentrations of CLSE added to the incubation medium after preformation of the fibrin matrix did not substantially influence the kinetics of fibrinolysis. CLSE incorporation into the nascent fibrin clot resulted in complete loss of surface activity, but adsorption and surface tension-lowering properties were largely restored by subsequent plasmic clot lysis. Arising fibrin split products were shown to display similar inhibitory strength on CLSE surface activity compared with fibrinogen split products.(ABSTRACT TRUNCATED AT 250 WORDS

Studies of human pituitary LH containing internally cleaved β subunit

1991 ◽  
Vol 6 (1) ◽  
pp. 101-109 ◽  
Author(s):  
A. Stockell Hartree ◽  
R. C. Shownkeen
Keyword(s):  
Receptor Binding ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peptide Bond ◽  
Β Subunit ◽  
Human Pituitary ◽  
Α Subunit ◽  
Peptide Bond Cleavage

ABSTRACT We have investigated the origin of the internal peptide bond cleavage found in the β subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between α and β subunits and on binding of hLH to its receptor. The content of cleaved β subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked β subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked β subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 °C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS). When hLH was incubated at 37 °C at pH 55 or pH 95 in the absence of enzyme inhibitors, some cleaved β subunit was found in the hLH—ANS dimer form of the hormone, but most of the nicked subunit appeared in fractions of lower molecular weight and with low receptor-binding activity. Fractions isolated as hLH—ANS dimer had receptor-binding activities which were negatively correlated with their content of cleaved β subunit, the most active fraction (21 000 i.u./mg) being virtually free of this component. Our studies suggest (1) that the cleavage is caused by proteolytic enzyme activity present in human pituitary tissue and in purified preparations of hLH, (2) that free hLH-β subunit is cleaved far more readily than when it is combined with α subunit in the intact hormone, (3) that the presence of the cleavage in free hLH-β prevents refolding of this subunit to the correct conformation that permits its combination with α subunit to regenerate native hormone, and (4) that the site of cleavage is likely to be at a peptide bond located on the surface of the intact hLH molecule.

scholarly journals Purification and Biochemical Characterization of the VIM-1 Metallo-β-Lactamase

2000 ◽  
Vol 44 (11) ◽  
pp. 3003-3007 ◽  
Author(s):  
Nicola Franceschini ◽  
Berardo Caravelli ◽  
Jean-Denis Docquier ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Metal Removal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Mediterranean Area ◽  
Exchange Chromatography ◽  
Amino Terminal ◽  
Chromatography Step

ABSTRACT VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla VIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106M−1 · s−1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

scholarly journals Deposition of kappa and lambda light chains in amyloid filaments of dialysis-related amyloidosis.

1995 ◽  
Vol 6 (4) ◽  
pp. 1262-1270
Author(s):  
D Brancaccio ◽  
G M Ghiggeri ◽  
P Braidotti ◽  
A Garberi ◽  
M Gallieni ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Extraction ◽  
Major Constituent ◽  
Light Chains ◽  
Staining Procedure ◽  
Immunogold Staining ◽  
Amino Terminal ◽  
Amyloid Deposits

beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

scholarly journals Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) fromAlcaligenes faecalis N-38A

1999 ◽  
Vol 65 (2) ◽  
pp. 712-717 ◽  
Author(s):  
Atsuhisa Nishimura ◽  
Yasunori Ozaki ◽  
Hiroshi Oyama ◽  
Takashi Shin ◽  
Sawao Murao
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alcaligenes Faecalis ◽  
Cell Extract ◽  
Homogeneous State ◽  
Amino Terminal ◽  
A Cell ◽  
Terminal Amino

ABSTRACT A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [l-glutamate]/[l-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

Plasmic degradation of fibrin rapidly decreases platelet adhesion and spreading

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1143-1150 ◽  
Author(s):  
M Hamaguchi ◽  
LA Bunce ◽  
LA Sporn ◽  
CW Francis
Keyword(s):  
Platelet Adhesion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fibrin Clot ◽  
Amino Terminus ◽  
Beta Chain ◽  
Gamma Chain ◽  
In Vitro System ◽  
Extensive Degradation

Abstract Plasmin cleaves fibrin at or near sites involved in platelet recognition and may modulate platelet adhesion and spreading. Using an in vitro system, we have characterized the effects of limited plasmic degradation of polymerized fibrin on platelet adhesion and spreading. As shown by scanning electron microscopy, exposure to plasmin changed the tight fibrillar fibrin surface to a less dense structure with irregular and broken fibers. There was a gradient of proteolytic degradation through the fibrin clot as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the most extensive degradation at the surface. Plasmic degradation resulted in a rapid and progressive decrease in platelet adhesion. Plasmin exposure for 5 minutes resulted in only 6% solubilization of the fibrin but a 56% decrease in platelet adhesion. After 30 minutes of plasmin exposure, spreading of adherent platelets on fibrin also decreased sharply to a minimum of 35% of baseline. Inhibition experiments with specific monoclonal antibodies (MoAbs) indicated that platelet adhesion to undergraded fibrin involved residues within the sequence 566 through 580 of the alpha chain (including the RGDS site), the carboxyl terminal dodecapeptide of the gamma chain, and the amino terminus of the beta chain. MoAb 7E3, reactive with alpha IIb beta 3, inhibited platelet adhesion to fibrinogen by 90% +/- 5%, and to desA fibrin, prepared with Reptilase (American Diagnostica, Greenwich, CT), by 94% +/- 6%, whereas inhibition of adhesion to undegraded desAB fibrin was significantly less (48% +/- 8%, P > .01). The addition of 7E3 to MoAb T2G1, reactive with beta 15–21, significantly increased inhibition to desAB fibrin to 69% +/- 6% (P < .025), suggesting that the newly exposed amino terminus of the beta chain contributes to platelet adhesion. The results show that plasmin exposure of fibrin markedly decreases platelet adhesion and spreading, suggesting that plasmin degradation may play a role in modulating cellular responses to fibrin.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

Sign in / Sign up

Export Citation Format

Share Document