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Biomedical subjects

J G Bieth

Publications and source records attributed to J G Bieth.

At least 73 records · Page 4Linked to original sources
PubMed1988

[Human leukocyte elastase].

This article reviews some properties of human leucocyte elastase. This 30 kDa glycoprotein formed of 218 amino acid residues, is a serine proteinase which cleaves proteins at Val-X, Ala-X, Leu-X or Met-X bonds. Leucocyte elastase solubilizes fibrous elastin and also degrades other extracellular matrix proteins. It hydrolyses and inactivates a number of plasma proteins. Synthetic substrates are more convenient than elastin to measure elastase activity. A large number of natural and synthetic inhibitors of leucocyte elastase have been described. The former include alpha 1-proteinase inhibitor or alpha 1-antitrypsin, inter-alpha-inhibitor, alpha 2-macroglobulin, bronchial and cervical mucous inhibitor and a number of animal and plant proteins. Numerous synthetic inhibitors with therapeutic potentials have been designed. The efficiency of an inhibitor depends, among others, upon its rate of association with the enzyme and upon the stability of the enzyme-inhibitor complex. Elastase probably plays a physiological function in neutrophil migration, phagocytosis and tissue remodeling. It apparently plays a pathological role in pulmonary emphysema, rheumatoid arthritis, infections and inflammation. The pathogenic role of leucocyte elastase is best understood in emphysema.

Blood Proteins↗
PubMed1987

Purification and characterization of human bronchial proteinase inhibitor.

We describe a purification procedure for the human bronchial proteinase inhibitor which involves trichloroacetic acid precipitation of sputum followed by ion-exchange and gel filtration chromatography. The inhibitor shows a major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but exhibits microheterogeneity on high-resolution chromatography. It has a molecular mass of 15.5-16 kDa as determined by electrophoresis and gel filtration and is 90% active against leukocyte elastase. The amino acid sequence of the N-terminal portion of the inhibitor was determined and was found to be identical (through 29 amino acids) to that recently reported for the human seminal plasma proteinase inhibitor I (Seemuller et al. (1986) FEBS Lett. 199, 43-48).

Amino Acid Sequence↗
PubMed1987

Fast solubilization of human lung elastin by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa may cause severe lung infections in humans. This bacteria secretes an elastase that might degrade lung elastin. We have studied the solubilization of human lung elastin by P. aeruginosa elastase in an attempt to delineate the pathogenic role of this proteinase in P. aeruginosa lung infections. We also used bovine ligamentum nuchae elastin and human leukocyte elastase for comparative purposes. With an elastin concentration of 5 mg X ml-1 and at physiologic ionic strength, P. aeruginosa elastase is about 50 times more active on human lung elastin than on bovine elastin. In contrast, human leukocyte elastase has similar specific activities on the 2 substrates. In addition, the bacterial enzyme is about 10 times more active on human elastin than the neutrophil elastase but the latter is about 5 times more active on bovine elastin than the former. In order to better quantitate these enzyme-substrate interactions, we have measured initial rates of elastolysis, derived from product versus time curves, as a function of elastin concentration. The substrate-velocity curves, analyzed using an equation similar to the classic Michaelis-Menten one, yielded 2 empirical kinetic parameters: [S50]-1, the apparent elastase-elastin affinity and Vm, the apparent catalytic efficiency of elastase. This analysis shows that human leukocyte elastase exhibits similar [S50]-1 and Vm values for the 2 elastins. The low activity of P. aeruginosa elastase on bovine elastin is due to the combined effects of low S50(-1) and Vm values, which could not be measured separately.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G.

We have investigated the inhibition of human leukocyte elastase and cathepsin G by recombinant Eglin c under near physiological conditions. The association rate constants k on of Eglin c for elastase and cathepsin G were 1.3 X 10(7) M-1 s-1 and 2 X 10(6) M-1 s-1, respectively. Under identical conditions, the k on for the association of human plasma alpha 1-proteinase inhibitor with the two leukocproteinases were 2.4 X 10(7) M-1 s-1 and 10(6) M-1 s-1, respectively. The consistency of these data could be verified using a set of competition experiments. The elastase-Eglin c interaction was studied in greater detail. The dissociation rate constant k off was determined by trapping of free elastase from an equilibrium mixture of elastase and Eglin c with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The rate of dissociation was very low (k off = 3.5 X 10(-5) s-1). The calculated equilibrium dissociation constant of the complex, Ki(calc) = k off/k on, was found to be 2.7 X 10(-12) M. Ki was also measured by adding elastase to mixtures of Eglin c and substrate and determining the steady-state rates of substrate hydrolysis. The Ki determined from these experiments (7.5 X 10(-11) M) was significantly higher than Ki(calc). This discrepancy might be explained by assuming that the interaction of Eglin c with elastase involves two steps: a fast binding reaction followed by a slow isomerization step. From the above kinetic constants it may be inferred that at a therapeutic concentration of 5 X 10(-7) M, Eglin c will inhibit leukocyte elastase in one second and will bind this enzyme in a "pseudo-irreversible" manner.

Cathepsin G↗
PubMed1987

The elastase inhibitory capacity and the alpha 1-proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects.

Pulmonary emphysema is currently thought to be due to an elastase-antielastase imbalance with resultant destruction of alveolar structures. The present study was aimed at testing whether alpha 1-proteinase inhibitor (alpha 1 PI) is the major component of the antielastase screen of the lower respiratory tract of healthy subjects. Bronchoalveolar lavage was performed in 8 nonsmokers (27.8 +/- 3.8 years) and 9 smokers (25 +/- 0.96 years). The lavage fluids were tested for leukocyte and pancreatic elastase inhibitory capacity (LEIC and PEIC) and immunoreactive alpha 1 PI and bronchial inhibitor (brI) content. The mean +/- s.e.m. levels of LEIC, PEIC, alpha 1 PI and brI were 0.16 +/- 0.039, 0.042 +/- 0.006, 0.09 +/- 0.007 and 0.013 +/- 0.002 mol/mol albumin, respectively. Thus, on the average, the molar concentration of brI was about 14% that of alpha 1 PI. The difference between LEIC and alpha 1 PI did not reach statistical significance (P = 0.0503). The PEIC was however significantly lower than the alpha 1 PI levels (P less than 0.05), indicating that the lavage fluids contained both active and inactive alpha 1 PI. Nonsmokers and smokers did not differ in their LEIC, PEIC, alpha 1 PI and brI levels. When the data were examined on an individual basis, the subjects could be divided into 2 groups: group I (n = 9; 3 nonsmokers, 6 smokers) whose LEIC/alpha 1 PI molar ratios were higher than unity and group II (n = 8; 5 nonsmokers, 3 smokers) whose LEIC/alpha 1 PI molar ratios were equal or lower than unity. Group I subjects had significantly higher LEIC values (0.26 +/- 0.05 mol elastase inhibited/mol albumin) than group II individuals (0.055 +/- 0.006; P less than 0.001) but the two groups had similar levels of immunoreactive alpha 1 PI (0.09 and 0.08 mol alpha 1 PI/mol albumin for group I and II, respectively), functionally active alpha 1 PI (percentage of active alpha 1 PI: 53% and 37% for group I and II, respectively) and immunoreactive brI (0.016 and 0.010 mol brI/mol albumin for group I and II, respectively). These results suggested that the lavage fluids from group I contained significant amounts of undefined leukocyte elastase inhibitor(s). Gel filtration of a lavage fluid from group I showed that the undefined elastase inhibitor(s) co-eluted with brI. Most of the lavage fluids were still able to inhibit leukocyte elastase following removal of alpha 1 PI by perchloric acid precipitation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1986

Influence of elastin on the inhibition of leucocyte elastase by alpha 1-proteinase inhibitor and bronchial inhibitor. Potent inhibition of elastin-bound elastase by bronchial inhibitor.

We have investigated the effect of human lung elastin on the inhibition of human leucocyte elastase by human alpha 1-proteinase inhibitor and bronchial inhibitor. Elastin was unable to dissociate the elastase-inhibitor complexes during the 150 min of the elastolysis reaction. When elastase was added to mixtures of elastin and alpha 1-proteinase inhibitor, it was fully bound to the latter. The competition between elastin and bronchial inhibitor was also in favour of the latter, but a 1.5 molar excess of inhibitor over elastase was required to achieve total binding of the enzyme. About 25% of elastin-bound elastase was found to be resistant to the inhibitory effect of alpha 1-proteinase inhibitor. The major isoenzyme and the mixture of the three minor isoenzymes of elastase exhibited similar behaviour. By contrast, bronchial inhibitor was as efficient in inhibiting the elastin-bound elastase as it was in inhibiting the free enzyme. This inhibitor was also able to inhibit fully the fraction of elastin-bound elastase that was resistant to alpha 1-proteinase inhibitor. We also describe a rapid procedure for the isolation of gram quantities of alpha 1-proteinase inhibitor.

Blood Proteins↗
PubMed1985

Cigarette smoke decreases the rate constant for the association of elastase with alpha 1-proteinase inhibitor by a non-oxidative mechanism.

This paper describes a non-oxidative impairment of the biological function of alpha 1-proteinase inhibitor by cigarette smoke. Aqueous solutions of cigarette smoke are able to decrease the rate constant kass for the inhibition of porcine pancreatic elastase by human plasma alpha 1-proteinase inhibitor. The value of kass decreases linearly with the concentration of smoke (from 2.2 X 10(5) M-1 s-1 to 0.6 X 10(5) M-1 s-1). This effect is not due to an oxidation of the inhibitor. When pancreatic elastase is reacted with elastin in the presence of alpha 1-proteinase inhibitor and cigarette smoke solution, elastolysis occurs at a rate nearly identical to that observed in the absence of inhibitor. This effect is due to a smoke-induced decrease in kass. These observations may serve as a model of biological regulation of proteolysis via a change in the rate constant for a proteinase-proteinase inhibitor association. The influence of cigarette smoke on the inhibition of human neutrophil elastase by alpha 1-proteinase inhibitor could not be studied in detail because the enzyme precipitates in the presence of concentrated smoke solution.

Animals↗
PubMed1985

Evidence that Pseudomonas aeruginosa elastase does not inactivate the bronchial inhibitor in the presence of leukocyte elastase. Studies with cystic fibrosis sputum and with pure proteins.

Pseudomonas aeruginosa elastase has recently been shown to inactivate bronchial inhibitor, the major leukoproteinase inhibitor of the bronchial tree. To test if this in vitro finding is relevant to pathology, we have measured the leukocyte elastase inhibitory capacity and the immunoreactive levels of bronchial inhibitor in the acidified and neutralized sputum specimens from 15 patients with cystic fibrosis, 11 of whom were infected by P. aeruginosa and 10 of whom exhibited free bacterial elastase activity. The percentage of functionally active bronchial inhibitor in these acid-treated sputum specimens (i.e., concentration of active inhibitor/concentration of immunoreactive inhibitor X 100) was found to be 125 +/- 17%. It was positively correlated with the free leukocyte elastase concentration (r = 0.77, p less than 0.001) but not with the free bacterial elastase concentration (r = 0.46, p greater than 0.05). Therefore, in an in vivo situation, P. aeruginosa elastase does not necessarily inactivate the bronchial inhibitor. Experiments using pure proteins show that the inactivation process does not take place when leukocyte and P. aeruginosa elastase are added simultaneously to the inhibitor. This suggests that the bronchial inhibitor reacts preferentially with leukocyte elastase and that in its complexed form it is shielded from the inactivating action of P. aeruginosa elastase. In addition, the inhibitor recovered from the acid-induced dissociation of the leukocyte elastase-inhibitor complexes, exhibits a substantially higher specific activity than does the native molecule. This explains why the average functional activity of the bronchial inhibitor is significantly higher than 100% in sputum samples from patients with cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1985

Smoking does not reduce the functional activity of serum alpha-1-proteinase inhibitor. An epidemiologic study of 719 healthy men.

The present study was aimed at testing the hypothesis that smoking, the major risk factor for the development of pulmonary emphysema, impairs the functional activity of alpha 1-proteinase inhibitor (alpha 1-antitrypsin). We used a population of 719 apparently healthy subjects. The serum concentrations of immunoreactive and functionally active alpha 1-proteinase inhibitor were measured by radial immunodiffusion and inhibition of porcine pancreatic elastase, respectively. Both the immunoreactive and functionally active levels of serum alpha 1-proteinase inhibitor were found to increase with tobacco consumption, but the ratio between the 2 concentrations was independent of smoking. Smoking does not, therefore, impair the functional activity of alpha1-proteinase inhibitor.

Adult↗
PubMed1985

The concentration of leukocyte elastase-alpha 1-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy human subjects.

Although alpha 1-proteinase inhibitor (alpha 1-antitrypsin) is widely thought to protect lung elastin against the elastolytic action of leukocyte elastase, there is only circumstantial evidence for such a protective role. We have demonstrated and quantified elastase-alpha 1-proteinase inhibitor complex in bronchoalveolar lavage fluids from healthy smokers and nonsmokers using a new enzyme-linked immunosorbent assay. The relative concentration of complex is 0.36 +/- 0.48 mmol/mol albumin in nonsmokers and 0.33 +/- 0.29 mmol/mol albumin in smokers. Less than 1% of lavage fluid alpha 1-proteinase inhibitor is complexed with elastase (0.31% in nonsmokers and 0.34% in smokers). This proportion is, however, much higher than in normal plasma where only approximately 0.006% of inhibitor is bound to elastase. Our data confirm that alpha 1-proteinase inhibitor efficiently acts as an antielastase barrier in the lower respiratory tract.

Bronchi↗
PubMed1985

The antielastase screen of the lower respiratory tract.

The protease-antiprotease theory of pulmonary emphysema holds that alveolar structures may be destroyed by neutrophil elastase but are normally protected from destruction by elastase inhibitors. Bronchoalveolar lavage allows to collect three types of antielastases: alpha 1-proteinase inhibitor (alpha 1 PI), bronchial mucus inhibitor and "unidentified inhibitors". Alpha 1 PI acts as an irreversible inhibitor of serine-proteinases. It reacts much faster with neutrophil elastase than with other enzymes and is therefore considered as a physiological inhibitor of this leukoproteinase. Oxidation of Met into methionine sulfoxide leads to a dramatic reduction of the inhibitory capacity of alpha 1 PI. This oxidative inactivation may be brought about by oxidants excreted by phagocytes and cigarette smoke condensate. A back-up control is provided by methionine sulfoxide reductase, an enzyme present in phagocytes and capable of reducing oxidized alpha 1 PI. The bronchial inhibitor is an acid-stable protein secreted by the mucus cells of the respiratory tract. It is a reversible tight-binding inhibitor which reacts with neutrophil elastase. Although it occurs in low amounts in the lower respiratory tract it may play an active physiological role because of its low molecular weight which allows its easy diffusion within the interstitial tissue.

Blood Proteins↗
PubMed1984

A simple spectrophotometric assay of plasminogen activator: comparison with the fibrinolytic method.

We describe a simple assay of plasminogen activator in which the enzyme reacts with a mixture of plasminogen and H-D-valyl-L-leucyl-L-lysine-p-nitroanilide for 1 h at 37 degrees C after which the absorbance is measured at 405 nm. The method detects as little as 2 CTA milliunits of activator and is linear over a 100-fold range of enzyme concentration. The new procedure has been used successfully for the assay of activator in breast tumor cytosols, cell culture supernatants, and pleural or ascitic fluids. Thirty-two biological samples have been assayed for plasminogen activator activity with both the spectrophotometric method and a classical fibrinolytic technique using radiolabeled fibrin. Although the two series of results are significantly correlated, the activities measured with the former assay are significantly different from those determined with the latter. It is shown that the spectrophotometric method is, in many respects, superior to the fibrinolytic procedure.

Ascitic Fluid↗
PubMed1984

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor.

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.

Benzamidines↗
PubMed1984

Temperature and pH dependence of the association rate constant of elastase with alpha 2-macroglobulin.

Binding kinetics of porcine pancreatic elastase to human alpha 2-macroglobulin was monitored by measuring the enzymatic activity of alpha 2-macroglobulin-bound elastase on succinyltrialanine p-nitroanilide after inhibition of free elastase by alpha 1-proteinase inhibitor. The association of the two proteins follows second-order kinetics with a rate constant ka = 4.4 X 10(6) M-1 S-1 at pH 8.0 and 25 degrees C. The rate of association strongly increases between pH 5.0 and 8.0, suggesting that the rate-limiting step of binding is the proteolytic cleavage at the bait region of the macroglobulin. The study of the temperature dependence of ka shows that the binding of elastase to alpha 2-macroglobulin is characterized by a positive entropy of activation (delta S* = +35.3 e.u. at 25 degrees C).

Animals↗
PubMed1984

Arginine modification in elastase. Effect on catalytic activity and conformation of the calcium-binding site.

Chemical modification of 2 +/- 0.5 arginine residues of porcine pancreatic elastase by 1,2-cyclohexanedione leads to an 85 +/- 5% loss of activity with the specific substrate N-succinyltrialanine p-nitroanilide. Modification of additional arginines does not completely abolish the enzyme activity. The modification reaction is very fast (second order rate constant = 0.24 M-1 S-1) and involves only arginine residues. Acetyltetraalanine or trifluoroacetyltetraalanine decreases the rate of cyclohexanedione-induced inactivation of the enzyme but does not significantly change the number of modified arginine residues. Other dicarbonyl reagents, butanedione or phenylglyoxal, also react with elastase but at much lower rates. Cyclohexanedione-modified elastase is partially active against a series of synthetic substrates of varying chain length. The partial inhibition results from a 2- to 5-fold increase in Km while kappa cat is increased for most substrates. For N-succinyltrialanine p-nitroanilide both the acylation and deacylation rate constants are decreased. The Ki values of a series of acetylated and trifluoroacetylated inhibitors increase 2- to 5-fold. Modified elastase is still able to react with fibrous elastin and with plasma alpha 1-proteinase inhibitor but at significantly lower rates. Modification of one arginine residue alters the properties of the calcium-binding site of elastase as demonstrated by terbium luminescence experiments. The affinity of enzyme for terbium is decreased by a factor of 10 and the circularly polarized luminescence spectrum of the terbium-elastase complex is considerably flattened. Modification of further arginine residues does not increase the extent of these alterations. Circular dichroism shows that the overall conformation of elastase is not altered following arginine modification. We speculate that the two residues modified by cyclohexanedione are Arg 65, located at about 8 A from the metal ion-binding site, and Arg 217A, located at the S'3 subsite of elastase.

Animals↗