elastin and succinyl-trialanine-4-nitroanilide

Experimental immunization of rabbits with soluble porcine elastin was performed. Elastase-like activities in plasma and the aorta were assayed using succinyl trialanine p-nitroanilide. A statistically significant increase in the elastase-like activity in plasma of the immunized animals was observed. Estimations of the elastase-like activity in the rabbit aorta showed the rise of analyzed parameters in the experimental group.

Topics: Animals; Aorta; Elastin; Male; Oligopeptides; Pancreatic Elastase; Rabbits

Fragmented elastic fibers are known to be associated with atherosclerotic lesions, angitis and age-related changes of the vessel wall. We produced fragmented elastic fibers in the thoracic aorta by immunizing rabbits with elastin peptides. Fragmented elastic fibers in the media were associated with degenerative smooth muscle cells, and the number of fine elastic fibers beneath the endothelium was decreased. The lesion presented here resembled age-related changes of the vessel wall. We also evaluated the influence of elastase administration on the formation of this lesion. In rabbits receiving elastin immunization with simultaneous intraperitoneal administration of elastase, fragmented elastic fibers were apparently decreased and the appearance of the elastic fibers resembled that of normal rabbits. These data suggest the possibility that administered elastase blocks the formation of fragmented elastic fibers in the aortic wall.

Topics: Animals; Antibodies; Aorta, Thoracic; Aortic Diseases; Elastin; Hydrolysis; Immunization; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Oligopeptides; Pancreatic Elastase; Peptides; Rabbits

Topics: Cations, Divalent; Cells, Cultured; Cutis Laxa; Elastin; Fibroblasts; Humans; Keratosis; Oligopeptides; Pancreatic Elastase; Pseudoxanthoma Elasticum; Skin

We have determined the effect of two elastase-specific synthetic low molecular weight substrates, L-pyroglutamylprolylvaline-paranitroanilide and succinyltrialanyl-paranitroanilide, together with insoluble elastin-fluorescein, on the determination of the neutrophil elastase (NE) inhibitory capacity of purified alpha 1-proteinase inhibitor (alpha 1-PI) and bronchial antileukoprotease (ALP). In addition, the inhibitory capacities of mixtures of alpha 1-proteinase inhibitor, antileukoprotease and alpha 2-macroglobulin prepared in ratios similar to that in lung secretions were determined. Purified inhibitors, alone or in combinations, inhibited about 1 mole neutrophil elastase per mole inhibitor when assessed using synthetic substrates. However, when elastin-fluorescein was used in the assay system, the purified inhibitors showed an inhibitory capacity that was 40-85% of the value obtained using synthetic substrates. Even less inhibition was observed when mixtures of inhibitors were assessed using elastin-fluorescein (23-44% of the value for synthetic substrates). Our data indicate that results of elastase inhibitor activity measurements depend on the type of substrate which has been used.

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Blood Proteins; Bronchi; Bronchitis; Chronic Disease; Elastin; Humans; Molecular Weight; Neutrophils; Oligopeptides; Pancreatic Elastase; Protease Inhibitors; Proteinase Inhibitory Proteins, Secretory; Proteins; Pyrrolidonecarboxylic Acid

Fibroblasts from normal adult forearm skin and neonatal foreskin were cultured and examined for their ability to synthesize and secrete elastase and neutral cathepsin. All of the cultures examined produced detectable amounts of elastase using insoluble elastin as substrate. An enzyme was also found that hydrolyzed the synthetic elastin substrate, N-succinyl-(Ala)3-p-nitroanilide, but did not degrade insoluble elastin. In addition, activity against the synthetic cathepsin substrate N-benzoyl-DL-phenylalanine-naphthyl ester was found. Inhibitor profiles indicate that the elastin and N-succinyl-(Ala)3-p-nitroanilide degrading activities are due to metalloproteinases. Degradation of N-benzoyl-DL-phenylalanine-naphthyl ester can be inhibited by phenylmethylsulfonyl fluoride. These proteinases were usually found associated with the cell layer. Although activities of the measured proteinases were detected in all cultures, increased or decreased enzyme activities were not predictably related to passage number or length of serum starvation. Degree of confluence also affected proteinase activities. Separation of the dermal-epidermal junction can be produced by the injection of these proteinases into intact mouse skin.

Topics: Adult; Cathepsins; Cells, Cultured; Elastin; Fibroblasts; Humans; Infant, Newborn; Male; Oligopeptides; Pancreatic Elastase; Peptide Hydrolases; Phenylalanine; Protease Inhibitors; Skin

Although the human alveolar macrophage in tissue culture can secrete an elastolytic metalloenzyme that is not inactivated by alpha 1-antiprotease (AAP), levels of this proteolytic activity and its relationship to AAP in human lung lavage fluid ( HLF ) are unknown from previous studies. Therefore, we measured elastolytic activity in concentrated (20- to 30-fold) HLF from 15 smokers and 10 nonsmokers and related results to measurements of AAP in these fluids. Activity (mean +/- SEM) against a C elastin substrate (expressed as nanograms of porcine pancreatic elastase equivalents per milligram of lavage fluid protein) in smokers, 18.9 +/- 6.7, significantly exceeded (p = 0.05) levels present in nonsmokers, 4.4 +/- 1.8. With the synthetic elastin-like chromophore substrate succinyl-trialanine-nitroanilide ( SLAPN ), activity in individual samples was reduced 79% by EDTA, a metalloproteinase inhibitor, whereas activity was reduced by only 29% in the presence of PMSF, a serine proteinase inhibitor. In addition, using a pooled sample of HLF and C elastin substrate, 80% of activity against the elastin substrate was eliminated by EDTA, whereas 51% was eliminated by PMSF. The activity measured with C elastin substrate correlated inversely with antigenic AAP (r = -0.05, p = 0.01), but no correlations were found between this activity and HLF cell number, cell viability, differential count, or subject smoking history. The detection of activity with C elastin in HLF , with primarily a metalloenzyme inhibitor profile, in the presence of antigenically detectable AAP, may have pathogenetic relevance for emphysema in humans.

Topics: Adult; alpha 1-Antitrypsin; Edetic Acid; Elastin; Female; Humans; Lung; Male; Oligopeptides; Pancreatic Elastase; Protease Inhibitors; Pulmonary Alveoli; Smoking; Therapeutic Irrigation

Human serum was found to contain enzyme activities hydrolyzing succinyl trialanine paranitroanilide and 3H-kappa-elastin Sepharose substrates. Both types of activities could be partly abolished by serine active site titrants (phenylmethanesulfonylfluoride, diisopropylphosphorofluoridate) and partly by neutral chelating agents (EDTA; 1-10-phenanthroline). The combination of phenylmethanesulfonylfluoride and EDTA gave a complete inhibition of human serum elastase-type activities indicating the presence of at least two different types of elastases (serine and metalloproteases) in human serum. In nonsmokers, the average serum elastase-type activity on succinyl trialanine paranitroanilide was found equal to 78.1 ng/ml porcine pancreatic elastase equivalents and on 3H-kappa-elastin sepharose beads equal to 688.8 ng/ml. No statistically significant differences were observed in elastase levels in the sera of individuals presenting clinical symptoms of atherosclerosis. The sera of patients suffering from chronic obstructive lung diseases contained, however, higher amounts of elastase-type activities, respectively equal to 237.2 ng/ml on succinyl trialanine paranitroanilide and 1,096 ng/ml on 3H-kappa-elastin Sepharose beads and was quantitatively significant when compared with control subjects.

Topics: Adult; Aged; Arteriosclerosis; Elastin; Humans; Lung Diseases, Obstructive; Middle Aged; Oligopeptides; Pancreatic Elastase; Sepharose; Substrate Specificity

The effect of some divalent cations on the elastolysis of insoluble elastin by pancreatic elastase has been studied with a conductimetric method. Mn2+ and Cu2+ give progressive inhibition, while Ca2+ initially slightly increases elastolysis, then becomes an inhibitor at concentrations higher than 10(-4) M. A stronger effect is demonstrated with Mg2+ which increases the initial velocity of the enzymatic reaction. However, there is no effect when N-succinyl-(Ala)(3)-paranitroanilide is used as the substrate. The different effects observed can be explained by an enzymatic activation process (cation-enzyme complex) and/or by a conformational change of the insoluble elastin.

Topics: Anilides; Animals; Calcium; Cattle; Copper; Dose-Response Relationship, Drug; Elastin; Electric Conductivity; Hydrolysis; Magnesium; Manganese; Oligopeptides; Pancreatic Elastase

The enzymatic degradation of insoluble elastin has been studied at several pH values using purified pepsin and cathepsin D, and neutrophil extracts. Pepsin degraded elastin throughout the pH range of 1.2-4.0 with the optimum pH below 2.0. Molecular sieve chromatography and gel electrophoresis indicated that a spectrum of molecular weight degradation products was produced. The degradation by pepsin was inhibited by sodium dodecyl sulfate (SDS), NaCl and pepstatin. Cathepsin D, which, like pepsin, degrades hemoglobin at acid pH and is inhibited by pepstatin, had no activity against insoluble elastin in the pH range of 3.2-7.2. Extracts of neutrophils degraded elastin above pH 4.0. The pH profile of elastin degradation by neutrophil extracts generally followed that of purified human leukocyte elastase. Our results suggest that during alimentation or pulmonary aspiration of gastric contents, extracellular elastin may be digested by gastric juice at acid pH. Inflammatory cells would not appear to be capable of contributing to such actions until local pH approaches neutrality. Cathepsin D, a major constituent of inflammatory cells, does not digest all types of connective tissue proteins.

Topics: Anilides; Animals; Cathepsin D; Cathepsins; Cattle; Chromatography, Gel; Elastin; Hydrogen-Ion Concentration; Methemoglobin; Neutrophils; Oligopeptides; Pepsin A; Swine

Topics: Anilides; Animals; Elastin; Humans; Kinetics; Neutrophils; Oligopeptides; Osmolar Concentration; Pancreas; Pancreatic Elastase; Sodium Chloride; Swine