Antiproteinase Activity Research Articles

Proteinases and restenosis in the human coronary artery: extracellular matrix production exceeds the expression of proteolytic activity

To understand the balance of proteinase antiproteinase activity and the production of extracellular matrix (ECM) at the site of arterial injury, we analyzed the composition of ECM and proteinase activity in normal internal mammary arteries, tissue samples obtained from atherosclerotic coronary lesions and restenotic lesions obtained during directional coronary atherectomy. Histologically and biochemically, collagen and proteoglycans increased, and elastin decreased in samples from restenotic lesions when compared to samples taken from patients undergoing their first revascularization (de novo). In contrast, cellularity was increased in samples obtained from de novo patients as compared to samples obtained from restenotic lesions. Intrinsic activity of matrix metalloproteinases (MMPs) was measured by using zymography and scanning all the lytic bands in zymographic gel. In these gels, identical amounts of total protein were loaded in each lane. MMP activity was determined as % of the total (latent and active) MMPs after trypsin activation (100%) in the normal artery. Intrinsic MMP activity was reduced to 6% ± 1% in atherosclerotic lesions and 1% ± 1% in restenotic lesions, when compared to activity found in normal (10% ± 3%) arteries. Based on solubilization of fluorescein-conjugated elastin by the extracts, the MMP-mediated elastinolytic activity was 0.2 ± 0.1, 8.8 ± 1.5, and 24.0 ± 3 nmol/min/mg in restenotic, native atherosclerotic and normal tissue, respectively. The results suggested that, in arterial tissue from patients with angiographic restenosis, there is an increased production of ECM collagen and a decrease in MMP activity compared to both normal artery and atherosclerotic samples from de novo patients undergoing an initial revascularization procedure of a significant coronary artery lesion.

GUT PROTEINASE ACTIVITY IN INSECT PESTS OF CANOLA

AbstractThe digestive proteinases of three important pests of canola,Brassica napusL. andB.rapaL., in western Canada were characterized by assessing the proteolytic activity of homogenates of their midguts against azocasein or azoalbumin at various pH levels and in the presence of diagnostic proteinase inhibitors. The midgut of larvae of the bertha armyworm,Mamestra configurataWlk., had maximum proteolytic activity at pH 10.5 which was inhibited 45–60% by serine proteinase inhibitors such as the soybean trypsin inhibitor. The midgut of larvae of the diamondback moth,Plutella xylostellaL., had maximum proteolytic activity at pH 10 which was inhibited 56–75% by serine proteinase inhibitors. The two lepidopterans thus use a serine-like proteinase in digestion. The midgut of adults of the flea beetle,Phyllotreta cruciferaeGoeze, exhibited maximum proteolytic activity at pH 5 which was inhibited 33–61% by specific cysteine proteinase inhibitors such as cystatin andtrans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane (E-64) and was activated strongly byL-cysteine. Aspartic proteinase inhibitors such as pepstatin A also decreased proteolytic activity by 21–50%. Serine proteinase inhibitors were without effect. Therefore,P.cruciferaeappears to use both cysteine- and aspartic-like proteinases in digestion. Cotyledons and first true leaves of canola,B.napuscv. Westar, contained inhibitory activity against serine, cysteine, and aspartic proteinases when tested against bovine trypsin, papain, or porcine pepsin, but the level of antiproteinase activity is insufficient to provide significant resistance against any of these pests.

Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1-8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activity in vitro when they were incubated with hydrogen peroxide.

Quartz inactivates alpha 1-antiproteinase: possible role in mineral dust-induced emphysema.

Occupational exposure to some types of mineral particles has been shown to be associated with the development of emphysema, but the mechanism of this process is unknown. Because many mineral particles are known to catalyze the formation of active oxygen species in aqueous solution, we hypothesized that mineral particles could oxidatively inactive antiproteinases, leading to an imbalance between protease and antiprotease activities, events similar to those believed to occur with cigarette smoke. To test this hypothesis, human alpha 1-antiproteinase (alpha 1-AP) was incubated with suspensions of freshly ground or aged quartz, and antiproteolytic activity was determined by using porcine pancreatic elastase. Increasing concentrations of quartz were associated with increasing losses of antiproteolytic activity; this effect could be prevented by catalase. Freshly ground quartz was more active than aged quartz. Western blot analysis for alpha 1-AP showed abnormal banding, suggesting that porcine pancreatic elastase-alpha 1-AP complex formation was impaired by silica exposure. Chemical assay of aqueous quartz suspensions demonstrated production of hydrogen peroxide; incubation of alpha 1-AP with hydrogen peroxide caused a dose-dependent loss of antiproteolytic activity, and this also could be prevented by catalase. We conclude that, at least in vitro, quartz can inactivate alpha 1-AP through a hydrogen peroxide-mediated mechanism and that oxidative loss of antiproteinase activity could play a role in mineral dust-induced emphysema.

The inflammatory and proliferative response of normal skin in a model for acute chemical injury: ornithine decarboxylase induction as a common feature in various models for acute skin injury

Application of sodium dodecyl sulphate (SDS) on the skin of healthy volunteers was used as a model for acute chemical injury. The time course of the response with respect to cell proliferation was studied using ornithine decarboxylase (ODC) activity. Erythema, polymorphonuclear leucocyte (PMN) infiltration, and the induction of epidermal antiproteinase activity (SKALP/elafin) were used as markers for the inflammatory response. ODC induction was similar to that in other models of acute skin injury, such as tape-stripping and ultraviolet light radiation. The amount of PMN infiltration correlated with erythema, but not with ODC induction. In contrast with findings in the tape-stripping model, no induction of SKALP/elafin activity was found after SDS application. We conclude that cell proliferation as measured by ODC induction is a common feature in the various models for skin injury. Both the kinetics and the intensity of the inflammatory response, and the induction of epidermal antiproteinase activity, appear to vary, depending on the specific model.

The interaction of hydrophobic bile acids with the α 1-proteinase inhibitor

An in vitro complex formation between cholesterol and human α 1-proteinase inhibitor (α 1-antitrypsin, α 1-Pi) has been described. Hydrophobie bile acids were studied for a similar interaction using lithocholic acid (LC) as a prototype of a hydrophobic acid. At a molar ratio of 5:1, LC induced conformational changes of α 1-Pi reflected in an abnormal gel-electrophoretic appearance, loss of anodal immunoreactivity on crossed immunoelectrophoresis, exposition of new antigenic determinant(s) on immunodiffusion, and loss of antiproteinase activity. After 6 h incubation, LC and α 1-Pi form a complex of approximately 200 kDa molecular mass seen following gel-filtration. After prolonged (24 h) interaction a series of large α 1-Pi polymers were seen on SDS-PAGE under reducing conditions followed by Western blotting. Glycolitho-, sulfolitho-, deoxycholic and 3-β-hydroxy-5-cholenoic acids induced similar but less pronounced changes of α 1-Pi, whereas transferrin remained unaffected. Hydrophilic acids lacked effect on α 1-Pi. The results are compatible with a specific, irreversible interaction of α 1-Pi with hydrophobic bile acids affecting its physical and proteinase inhibitory properties. The cholestatic potency of the hydrophobic acids studied and their ability to induce α 1-Pi polymerization may be important in cholestatic conditions.

Selective inhibition of microbial serine proteases by eNAP-2, an antimicrobial peptide from equine neutrophils

Equine neutrophil antimicrobial peptide 2 (eNAP-2), a recently described antimicrobial peptide isolated from equine neutrophils, was found to selectively inactivate microbial serine proteases (subtilisin A and proteinase K) without inhibiting mammalian serine proteases (human neutrophil elastase, human cathepsin G, and bovine pancreatic trypsin). Although the primary structure of eNAP-2 resembled that of several known antiproteases that belong to the 4-disulfide core peptide family, this pattern of selectivity is unique. eNAP-2 formed a noncovalent complex with native subtilisin A or proteinase K but did not associate with these enzymes if they had been treated with phenylmethylsulfonyl fluoride, a serine protease inhibitor. The eNAP-2-microbial protease complex was disrupted by boiling or by exposure to low pH. We suggest that eNAP-2 exerted selective antiproteinase activity by binding tightly but noncovalently to the active site of subtilisin A or proteinase K. Since microbial exoproteases may act as virulence factors, the combined antimicrobial and antiprotease activities of eNAP-2 could allow it to play an important role in neutrophil-mediated antimicrobial defenses.

Characterization and gene sequence of the precursor of elafin, an elastase-specific inhibitor in bronchial secretions.

Human bronchial mucous secretions have been shown to contain inhibitors of serine proteinases secreted by neutrophils. The role of these inhibitors is probably to control the enzymes secreted in the airways and in the lung interstitium. Three of these inhibitors have been identified and characterized: alpha 1-proteinase inhibitor, mucus proteinase inhibitor, and elafin. The elafin molecule, a 6.0 kD inhibitor of serine proteinases shows homology with mucus proteinase inhibitor. We recently isolated both molecules in bronchial secretions. In this report, we present evidence for the existence of a precursor of the elafin molecule. We have cloned and sequenced the gene for this precursor and show that it is composed of three exons. The coding information for a 117 amino acid precursor protein of elafin (inclusive of the signal peptide) is contained in the first two exons. This was confirmed at the mRNA and protein levels. By Northern Blot analysis we detected a 800 bp long product, and by immunoaffinity we detected in sputum and in cultured epithelial cell supernatant (NCI-H322 cell line) a 12 kD protein species cross-reacting with anti-elafin IgG. The finding of possible cross-linking function for the precursor in addition to its antiproteinase activity indicates a possible role for this molecule as a cross-linker agent in the extracellular matrix.

In vitro complex formation between cholesterol and α 1-proteinase inhibitor

The in vitro interaction between human (α 1-proteinase inhibitor (α 1-PI) and cholesterol was studied with electrophoretic and gel Chromatographic methods. The addition of cholesterol (from 1 to 20 mol mol α 1-PI) at 37°C resulted in retarded electrophoretic mobility of α 1-PI towards the anode, diminished immunoreactivity and antiproteinase activity. At a molar ratio of 2:1 (cholesterol/α 1 -PI), antitryptic activity was reduced by 15% but antielastase activity by 50%. At this ratio the gel filtration α 1-PI peak appeared at 67 kDa, as compared to 52 kDa for native α 1-pi. No size difference was noted on SDS-PAGE. These results suggest the occurrence of noncovalent complex formation between cholesterol and α 1-PI in vitro.

Sera and leukocyte elastase-type protease and antiprotease activity in healthy and atherosclerotic subjects of various ages.

Serum and granulocyte elastase-type protease activities were determined simultaneously with their main plasma proteinase inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin in healthy control and atherosclerotic (ATS) subjects. The age-related associations of these parameters were also investigated. Serum elastase-type protease activity increased, but not statistically significantly, with aging in both control and ATS subjects. The enhancement of elastase-type protease activity in sera of ATS patients was significantly (p less than .02) greater than control subjects only in the case of the elderly. The granulocytes' elastase activity was significantly greater in granulocytes derived from both middle-aged and elderly ATS patients (p less than .03 and p less than .06) compared to age-matched control subjects. Alpha-1-antitrypsin was not significantly lower, whereas alpha-2-macroglobulin was significantly lower in sera of ATS subjects compared to age-matched control subjects (p less than .01). The conclusion is that increased elastase-type activity and decreased antiproteinase activity should be considered as potential factors in atherosclerotic arterial wall damage. The similarity of the results in the elderly and the ATS subjects suggest that atherosclerosis is an early aging process.

Antithrombin Budapest 3 An antithrombin variant with reduced heparin affinity resulting from the substitution L99F

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, C TC→ T TC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Antithrombin III Budapest: A Single Amino Acid Substitution (429Pro to Leu) in a Region Highly Conserved in the Serpin Family

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin-Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Decreased specific anti-elastase activity in the uninvolved skin of patients with psoriasis

Inhibitory activities against elastase, chymotrypsin and trypsin were studied in the fluid from experimentally developed suction blisters in the uninvolved skin of patients with psoriasis. These activities determined by spectrophotometry of specific synthetic low molecular weight substrates were compared with respective antiproteinase activities in sera of 32 patients with psoriatic lesions, ten patients in remission, and ten healthy volunteers. A marked reduction (29.2%) in the specific elastase inhibitory activity of blister fluid was found in patients with psoriasis when compared with normal subjects (p less than 0.05), since neither chymotrypsin nor trypsin inhibitory activities were altered. This reduction was despite about a 30% increase in the elastase inhibitory activity in the sera of these patients, which was related presumably to their increased activity of alpha 1-proteinase inhibitor, the main serum antiserine proteinase inhibitor. A decreased blister fluid:serum elastase inhibition ratio was shown in a large majority of patients with psoriasis, even in symptomless patients. The deficiency in specific elastase inhibitory activity of suction blister fluid was predominantly associated with early onset of psoriasis, guttate lesions and inactive lesions, skin involvement less than 20% of body surface, duration of relapse shorter than 2 months, and frequent relapses. These data indicate that the uninvolved skin of patients with psoriasis contains low concentrations of specific elastase tissue inhibitor, which deficiency might result in an excessive in vivo hydrolytic activity of neutrophil elastase released from migrating cells in the psoriatic skin.

48 ALPHA-1-PROTEINASE INHIBITOR (Pi) PHEHOTYPES IN CHILDREN WITH URINARY TRACT MALFORMATIONS

Pi phenotypes associated with decreased serum level of this protein are known to be more prevalent in subjects with a variety of diseases. The aim of this study was to reveal a possible linkage between deficient Pi phenotypes and urinary tract malformations. 374 pediatric patients were examined, including 72 with duplicated system, 97 with hydronephrosis, 23 with renal hypoplasia or aplasia, 179 with vesicoureteral reflux (VUR) and 3 with polycystic kidney disease. Pi typing was done by isoelectric focusing. Control group consisted of 1442 subjects examined in a population study. An increased frequency of heterozygous phenotyoes PiMZ, PiMS and PiMF was found in children with VUR grade 1 and 2 (PiMZ in 8.8%, PiMS in 5.295, PiMF in 2.6% of patients comparing with 3.5%, 3.3% and 0.4%, respectively, in the controls). These findings might suggest a certain role of moderately decreased antiproteinase activity in the appearance of low grade VUR (possibly via chronic inflammatory process).

Tumor Cell Growth Inhibition by Liposome-Encapsulated Aromatic Polyamidines

Apart from its antiproteinase activity, the aromatic polyamidine TAPP-Br [the bromo derivative of 1,3-di-(p-amidinophenoxy)-2,2-bis-(p-amidinophenoxymethyl)propane (TAPP-H)] is able to inhibit the in vitro growth of a variety of tumor cell lines, including human melanoma, and breast and kidney carcinoma. We have now shown that TAPP-Br can efficiently be encapsulated into egg phosphatidylcholine vesicles. When incorporated into these liposomes, the inhibitory effect of TAPP-Br is significantly enhanced compared with that of the free drug. Based on these promising results, a proposal is made for the delivery of this antiproliferative agent to tumor cells by using liposomes as the vehicle.

Plasmatic antiproteinase activity enhancement by insoluble functionalized polystyrene surfaces

Antithrombogenic functional polymer surfaces have been obtained by grafting heparin or by substituting insoluble polystyrene with sulphonate and/or amino acid sulphamide groups. Their heparin-like properties have been related to their catalytic effects on the antithrombin III —thrombin complex formation. Amongst these antithrombogenic surfaces, this study demonstrates that some insoluble amino acid sulphamide derivatives of polystyrene strongly potentiate heparin cofactor II, in addition to antithrombin III. In contrast, an insoluble polystyrene sulphonate and, to a lesser extent, an insoluble heparin copolymer, are better catalysts of antithrombin III. It is hypothesized that such different behaviours result from different conformations of the species adsorbed onto the surfaces. The conclusions support the possible use of such amino acid sulphamide groups to prepare antithrombogenic surfaces in contact with blood.

Tetra-amidines exhibiting anti-proteinase activity: effects on oriented migration and in vitro invasiveness of a Chinese Hamster cell line transfected with the activated human T24-Ha-ras-1 oncogene

In the present paper we have investigated the effects of aromatic tetra-amidines on attachment, oriented migration and in vitro invasiveness of the Chinese Hamster FH06T1-1 fibroblast lung cell line, transformed with the activated human T24-Ha-ras-1 oncogene. The FH06T1-1 cell line is tumorigenic in nude mice and displays growth properties and biological features clearly distinct from those of the FH06N1-1 cell line, obtained after transfection of the same fibroblast cells with the normal Ha-ras-1 proto-oncogene. Attachment, oriented migration and invasiveness were analysed by culturing the cells on a reconstituted extracellular matrix, composed of collagen IV, laminin, entactin and heparan sulphate proteoglycans. The results obtained demonstrate that oriented migration is performed only by FH06T1-1 cells and that tetra-benzamidines are effective inhibitors of oriented migration and “in vitro” invasiveness of these tumorigenic cells. These findings should encourage further studies on the possible antimetastatic effects of these antiproteinase tetra-benzamidines on experimental animals.

Purification and Characterization of Two Related but Distinct Metalloproteinase Inhibitors Secreted by Bovine Aortic Endothelial Cells

Two metalloproteinase inhibitors were purified from serum-free medium conditioned by bovine aortic endothelial cells. One of these inhibitors, with a molecular weight of 30,000-34,000 (reduced) is identified as tissue inhibitor of metalloproteinases; the second inhibitor has a molecular weight of 27,500 (reduced) and 20,400 (unreduced), is not recognized by an antiserum against bovine tissue inhibitor of metalloproteinases, appears unglycosylated, and has 51% identity with tissue inhibitor of metalloproteinases by NH2-terminal amino acid sequence analysis. This inhibitor has antiproteinase activities similar to those of tissue inhibitor of metalloproteinases, with inhibition of classical collagenase, type IV collagenase, and gelatinases but not trypsin, plasmin, or bacterial collagenase. Other properties shared with tissue inhibitor of metalloproteinases include trypsin sensitivity, acid and heat resistance, and inactivation by reduction-alkylation. The presence of these inhibitors in endothelial cells suggests that they may play important roles in protecting the integrity of the vascular basement membrane.

Effect of acute-phase proteinase inhibitors on chemotaxis of rat polymorphonuclear leukocytes in vitro.

Rat serum obtained at 24 h after subcutaneous injection of carrageenin significantly suppressed chemotaxis of rat polymorphonuclear leukocytes (PMNs) in vitro. alpha 2 Acute-phase macroglobulin (alpha 2APM), alpha 1 proteinase inhibitor (alpha 1 PI) and cysteine-proteinase inhibitors (CPIs) are present at high concentration in the 24-h serum and known as acute-phase reactants in rats. These acute-phase proteinase inhibitors were purified from inflamed rat serum or exudate and their effect on PMN chemotaxis was studied by Boyden's method in vitro. alpha 2 APM (4 mg/ml) significantly suppressed PMN chemotaxis while alpha 1M was without effect, though both alpha 2APM and alpha 1M had a similar anti-proteinase activity. The results suggest that alpha 2APM suppressed PMN chemotaxis through the mechanism unrelated to its anti-proteinase activity. On the other hand, alpha 1PI (1 and 3 mg/ml) slightly but significantly suppressed PMN chemotaxis, whereas CPI-1 and CPI-2 had no inhibitory effect. These results suggest that alpha 2APM and alpha 1PI play a role in suppression of PMNs infiltration into the inflammatory site in the late-phase of acute inflammation.