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Biomedical subjects

J G Bieth

Publications and source records attributed to J G Bieth.

At least 91 records · Page 5Linked to original sources
PubMed1984

In vivo significance of kinetic constants of protein proteinase inhibitors.

We describe the in vivo significance of the kinetic parameters which characterize the interaction between proteinases and protein proteinase inhibitors. Knowledge of the second-order association rate constant kass and in vivo inhibitor concentration allows the calculation of the delay time of inhibition, i.e., the time required for complete inhibition of a proteinase in vivo. The influence of biological substrates on the delay time is also analyzed. The extent of substrate breakdown during the delay time of inhibition may be computed from the various constants describing the proteinase/substrate/inhibitor interactions and the biological concentrations of proteinase and inhibitor. The in vivo partition of a proteinase between two inhibitors may be calculated if the kinetic parameters are known. We define a stability time for enzyme-inhibitor complexes as a minimal time during which the complexes may be considered as stable. This time is related to kdiss the dissociation rate constant of the reversible enzyme-inhibitor complex or to k, the breakdown rate constant of the complex formed with temporary inhibitors. The overall stability of the complex depends upon the ratio between the inhibitor concentration and Ki, the equilibrium dissociation constant of the complex. If this ratio is higher than 1000, a reversible inhibitor behaves like an irreversible one in vivo whatever the enzyme concentration.

Hydrolysis↗
PubMed1983

The functional activity of alpha 1-proteinase inhibitor in bronchoalveolar lavage fluids from healthy human smokers and non-smokers.

Cigarette smoking is an important risk factor for pulmonary emphysema. In vitro experiments document cigarette smoke-induced inactivation of alpha 1-proteinase inhibitor, a protein which is thought to protect the lung interstitium against the deleterious action of neutrophil elastase. To assess the relevance of this in in vitro findings, we have measured the functional activity of alpha 1-proteinase inhibitor collected by bronchoalveolar lavage from twenty healthy volunteers (10 heavy smokers and 10 non-smokers). The total inhibitor concentration was measured by radial immunodiffusion. The active inhibitor concentration was determined by virtue of its elastase inhibitory capacity. We used pure and active site-titrated porcine pancreatic elastase and a kinetic assay with succinyl-trialanine-p-nitroanilide in order to get reliable and accurate results. In smokers and in non-smokers the percentage of functionally active alpha 1-proteinase inhibitor in bronchoalveolar lavage fluid is 54 +/- 12 and 38 +/- 14%, respectively. This difference is not significant. Serum alpha 1-proteinase inhibitor is 100% active in both groups. Our data disagree with previous reports suggesting the presence of fully active alpha 1-proteinase inhibitor in bronchoalveolar lavage fluid of non-smokers and of partially active inhibitor in the lavage fluid of smokers.

Adult↗
PubMed1983

Mapping of the S' subsites of porcine pancreatic and human leucocyte elastases.

Trifluoroacetyl dipeptide anilides have been synthesized and used to map the S' subsites of porcine pancreatic elastase and human leucocyte elastase. A confident mapping of these subsites, at least for the porcine enzyme, was possible since the x-ray crystallographic study of its complex with CF3CO-Lys-Ala-NH-Ph-p-CF3 at a resolution of 2.5 A (Hughes, D. L., Sieker, L. C., Bieth, J., and Dimicoli, J. L. (1982) J. Mol. Biol. 162, 645-658) shows the CF3CO group at the S1 subsite and the dipeptide anilide bound at sites close to the S'1-S'3 subsites. Furthermore the effect of substitution was easy to investigate since these ligands are reversible competitive inhibitors of elastases, whose mode of binding to the porcine enzyme has been shown by nuclear magnetic resonance spectroscopy (Dimicoli, J. L., Renaud, A., and Bieth, J. (1980) Eur. J. Biochem. 107, 423-432) to be essentially unique and common to all CF3CO peptide anilides. The total number of S' subsites was found to be three for both enzymes. The individual subsites have the following specificities: subsite S'1, in porcine pancreatic elastase this subsite prefers Lys to Ala or Glu. In human leucocyte elastase this subsite is less specific; subsite S'2, in porcine pancreatic elastase, this subsite has a marked specificity for Ala. It accommodates bulkier residues with some difficulty. In human leucocyte elastase there is a remarkable specificity for Leu at this subsite; subsite S'3, in porcine pancreatic elastase, this subsite has a high aromatic specificity. In human leucocyte elastase there is no such affinity in S'3 but favorable local interaction exists. These specificities are examined on the basis of the coordinates of the CF3CO-Lys-Ala-NH-Ph-p-CF3 . porcine pancreatic elastase complex. Furthermore the different specificities of the S'2 subsite found in our own work and proposed by Atlas (Atlas, D. (1975) J. Mol. Biol. 93, 39-53) are briefly discussed.

Anilides↗
PubMed1983

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids.

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.

Antibody Specificity↗
PubMed1983

Inhibition of human leucocyte elastase by polynucleotides.

We have found that nanomolar range concentrations of transfer RNA inhibit human leucocyte elastase activity against synthetic or natural substrates. Titration curves give a stoichiometry of 9 +/- 1 elastase molecules inhibited per tRNA molecule. The interaction seems essentially electrostatic since similar inhibitions were measured with poly r(A) or calf thymus DNA, and because the increase in ionic strength leads to a decrease of inhibition. This observation appears to be specific of human leucocyte elastase since porcine pancreatic elastase, and both human and bovine chymotrypsins are not inhibited by tRNA.

Animals↗
PubMed1983

Structural arrangement of the proteinase binding sites in human alpha 2-macroglobulin.

Using singlet-singlet energy transfer measurements with labeled-chymotrypsin-alpha 2-macroglobulin complexes, we find that the two proteinase binding sites of alpha 2-macroglobulin are separated from each other by 44 A. The free thiol groups generated upon reaction of alpha 2-macroglobulin with trypsin or chymotrypsin react with thiopropyl Sepharose, indicating that they are located at the surface of the complexes. Singlet-singlet energy transfer experiments from labeled proteinases to labeled thiols of alpha 2-macroglobulin show that the thiol groups are in close contact with the proteinase molecules whether the latter are covalently or noncovalently bound to alpha 2-macroglobulin. In addition, they are remote from the association interface between the Mr = 360,000 halves of alpha 2-macroglobulin. Using the same approach we demonstrate that the active sites of chymotrypsin molecules are separated by a distance of at least 20 A from the thiols group of each alpha 2-macroglobulin subunit.

Binding Sites↗
PubMed1982

Separation of free and chymotrypsin-bound alpha 2-macroglobulin by affinity chromatography. Its use to demonstrate that the two chymotrypsin-binding sites of alpha 2-macroglobulin are equivalent and independent.

Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively eluted either with acetic acid or with 40% glycerol, pH 8.0. This is the first report of a preparative separation of free and proteinase-bound alpha 2-macroglobulin. Using this affinity chromatographic system, we were able to demonstrate that the two chymotrypsin binding sites of alpha-2-macroglobulin are equivalent and independent.

Animals↗
PubMed1982

Kinetics of the inhibition of leukocyte elastase by the bronchial inhibitor.

The rate constant for the association between human leukocyte elastase (EC 3.4.21.11) and human bronchial inhibitor has been determined by competition experiments with alpha 1-proteinase inhibitor. This constant (1.1.10(7) M(-1) . s(-1)) is 6-times lower than that for the association of leukocyte elastase and alpha 1-proteinase inhibitor. The latter inhibitor is able to dissociate the leukocyte elastase-bronchial inhibitor complex with a rate constant 1.3.10(-4) s-1. The equilibrium dissociation constant Ki of the complex is 1.2.10(-11) M. The physiopathological significance of these constants is discussed.

Bronchi↗
PubMed1982

[Proteases, antiproteases and pulmonary emphysema].

A deficiency of alpha 1 antiproteases is associated with severe and early emphysema. This emphysema can be experimentally produced in animals by endotracheal instillation of elastolytic proteases. Thus it would seem that emphysema is linked to an imbalance between proteases and antiproteases at the pulmonary level. This work studies the proteases, whose role in the genesis of emphysema is highly probable in view of the data in the literature (leukocyte elastase), disputed (macrophage elastase) or transitory (microbial elastases). We contrast the main agents capable of inhibiting these proteases (alpha 1 antiprotease and bronchial inhibitors) or of changing their activity (alpha 2 macroglobulins). The relative importance of these antiproteases is discussed in the light of studies made on bronchial secretions and bronchoalveolar lavage. These irritants may influence the protease - antiprotease equilibrium and favour the development of emphysema by increasing the proteases or decreasing the antiproteases. It appears that tobacco, as well as infection and anything which sets in motion the pulmonary phagocytes favour the liberation of leucocyte elastase. These attacks inactive the alpha 1 antiproteases in addition to the bronchial inhibitor. They may be recognized by a change in elastolytic and anti-elastolytic activity observed in bronchial secretions and in bronchoalveolar lavage (which is more disputed in the latter).

Animals↗
PubMed1981

Stimulation of the elastolytic activity of leukocyte elastase by leukocyte cathepsin G.

Human leukocyte cathespin G strongly stimulates the rate of solubilization of human lung elastin by human leukocyte elastase. For instance, the elastolytic activity of an equimolar mixture of elastase and cathepsin G is more than 5 times higher than that of elastase alone. Optimal stimulation occurs only if cathepsin G and elastase act simultaneously on elastin. Potentiation of leukocyte elastase digestion of lung elastin may also be brought about by bovine alpha-chymotrypsin. This enzyme is about half as efficient as cathepsin G. Stimulation of leukocyte elastase activity by cathepsin G is about 3 times less pronounced with bovine ligamentum nuchae elastin than with human lung elastin. On the other hand, the elastolytic activity of porcine pancreatic elastase is only enhanced by 20 to 30% by cathepsin. Therefore, maximal potentiation of elastolysis occurs with the lung elastin/leukocyte elastase system. The pathologic relevance of these findings is discussed.

Amino Acids↗
PubMed1981

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor.

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.

Animals↗