Search PubMed⌕ Search

Biomedical subjects

J G Bieth

Publications and source records attributed to J G Bieth.

At least 37 records · Page 2Linked to original sources
PubMed1995

Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor.

Commercial low molecular mass heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor, the predominant antielastase of lung secretions (Faller, B., Mély, Y., Gérard, D., and Bieth, J.G. (1992) Biochemistry 31, 8285-8290). To study the kinetic mechanism of this rate enhancement, we have isolated a 4.5-kDa heparin fragment from commercial heparin. This compound is fairly monodisperse as shown by analytical ultracentrifugation. It binds elastase and inhibitor with a 1:1 stoichiometry and an equilibrium dissociation constant of 3 and 210 nM, respectively. It also forms a tight complex with EI. Flow calorimetry shows that the inhibitor-heparin interaction is characterized by a large negative enthalpy change (delta H0 = -45.2 kJ mol-1) and a small entropy change (delta S = -23.7 J K-1 mol-1). Stopped-flow kinetics run under pseudo-first-order conditions ([Io] >> [Eo]) show that in the absence of heparin the inhibition conforms to a simple bimolecular reaction, [formula: see text] where, ka = 3.1 x 10(6) M-1 s-1, kd = 10(-4) s-1, and Ki = 33 pM, whereas in the presence of heparin, E and I react via a two-step mechanism, [formula: see text] where Ki* = 86 nM, k2 = 2.2 s-1, k-2 = 10(-3) s-1, and Ki = 37 pM. Thus, heparin increases both the rate of inhibition by promoting the formation of a high affinity EI* intermediate and the rate of EI dissociation. Since the dissociation is negligible in bronchial secretions where the inhibitor concentration is much higher than Ki, it may be concluded that heparin significantly potentiates the inhibitor's antielastase potential in vivo.

Amino Acid Sequence↗
PubMed1995

DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity.

DNA binds neutrophil elastase and mucus proteinase inhibitor as evidenced by affinity chromatography on elastase-Sepharose, inhibitor-Sepharose and DNA-cellulose. DNA is a potent hyperbolic inhibitor of elastase. The polynucleotide-enzyme complex is partially active on synthetic substrates and on elastin. DNA strongly increases kdiss and Ki for the inhibition of elastase by mucus proteinase inhibitor [formula: see text] The above effects are all salt-dependent. At physiological ionic strength, DNA is a potent inhibitor of the elastolytic activity of elastase and increases kdiss and Ki for the elastase-mucus proteinase inhibitor interaction 160-fold and 100-fold, respectively.

Cellulose↗
PubMed1995

Mapping the heparin-binding site of mucus proteinase inhibitor.

Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), the physiological antielastase of airways as a result of its binding with the inhibitor [Faller, B., Mély, Y., Gérard, D., & Bieth, J. G. (1992) Biochemistry 31, 8285-8290]. To explore the heparin-binding site of the inhibitor, we have modified the lysine and arginine residues of MPI and its isolated C-terminal domain by using 4-N,N-(dimethylamino)azobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) [Chang, J. Y. (1989) J. Biol. Chem. 264, 3111-3115] and (p-hydroxyphenyl)glyoxal (HPG) (Yamasaki, R. B., Vega, A., & Feeney, R. E. (1980) Anal. Biochem. 109, 32-40], respectively. The derivatizations were done in the absence and presence of a 4.5 kDa heparin fraction with a low degree of polydispersity. The effect of chemical modification of the inhibitors on their affinity for heparin was tested using two complementary procedures, one based on the ability of heparin to accelerate the inhibition of chymotrypsin by the inhibitors and the other exploiting the affinity of the inhibitors for immobilized heparin. Modification of a limited number of lysine and arginine residues in full-length MPI led to a 6-fold decrease in affinity for heparin. The presence of the polymer during the modification reactions significantly prevented this effect. Amino acid sequencing unambiguously identified the heparin-protected lysines as Lys 13 and Lys 87, located on the N-terminal and C-terminal domains of MPI, respectively. Heparin apparently protects mainly two arginine residues from modification by HPG.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.

Amino Acid Sequence↗
PubMed1995

Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenous protein inhibitors, cystatins.

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).

Animals↗
PubMed1994

Heparin protects cathepsin G against inhibition by protein proteinase inhibitors.

Cathepsin G, a cationic serine proteinase present in neutrophils and monocytes, is able to cleave biologically important proteins and may thus participate in tissue destruction during inflammation. Its activity is physiologically controlled by the fast-acting serpins, alpha 1-anti-chymotrypsin (ka = 5 x 10(7) M-1 S-1) and alpha 1-proteinase inhibitor (ka = 2.7 x 10(5) M-1 S-1). We have shown that cathepsin G forms a tightly bound 1:1 complex with a 5-kDa heparin fragment (Kd = 1.9 x 10-8 M). The partial enzymatic activity retained by this complex is inhibited extremely slowly by the above 68- and 53-kDa serpins. The activity of the complex is also virtually resistant to inhibition by eglin c, and 8-kDa non-serpin inhibitor. A detailed kinetic investigation showed that the inhibition of heparin-bound cathepsin G by the three proteins proceeded via a two-step mechanism. [formula: see text] The three inhibitors have widely different Ki* values (0.18-13 microM) (Ki* = k-1/k1). Their isomerization constants k2 are, however, all in the same range and their extremely low values (0.7-3 ms-1) account for the very low rate of cathepsin G inhibition. The second-order inhibition rate constants k2/Ki* were 4300, 700, and 52 M-1 S-1 for alpha 1-antichymotrypsin, alpha 1-antitrypsin, and eglin c, respectively, indicating that, if heparin is present in vivo, the two former physiological inhibitors will be unable to prevent cathepsin G-mediated proteolysis. Neutrophil elastase binds the 5-kDa heparin fragment with an affinity identical to that of cathepsin G. alpha 1-Proteinase inhibitor reacts, however, much faster with heparin-elastase (ka = 1.8 x 10(6) M-1 S-1) than with heparin-cathepsin G (k2/Ki* = 700 M-1 S-1).

Cathepsin G↗
PubMed1994

Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor.

N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.

Amino Acid Sequence↗
PubMed1993

Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by alpha 1-proteinase inhibitor.

Heparin decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (< or = 6 microM) the enzyme-inhibitor association conforms either to a simple bimolecular reaction (E+I kass-->EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I Ki*<==>EI* k2-->EI with Ki* > 0.4 microM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha 1-proteinase inhibitor interaction: it favors the association by decreasing Ki* but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-alpha 1-proteinase inhibitor complex.

Amino Acid Sequence↗
PubMed1993

Oxidized and Met358-->Leu mutated alpha 1-proteinase inhibitor as substrates of Pseudomonas aeruginosa elastase.

This paper investigates the catalytic activity of Pseudomonas aeruginosa elastase using the bait region of the alpha 1-proteinase inhibitor as a substrate. The bacterial enzyme cleaves the Pro357-Met358 bond of the wild-type inhibitor and the recombinant Met358 inhibitor and the Pro357-Leu358 bond of the recombinant Met358-->Leu inhibitor with kcat/Km values of 9 x 10(4) M-1 s-1, 1.4 x 10(5) M-1 s-1 and 3.5 x 10(5) M-1 s-1, respectively. In contrast, the N-chlorosuccinimide-oxidized inhibitor (Met351 and Met358 = methionine sulfoxides) is cleaved at the Glu354-Ala355 position with a significantly lower rate (kcat/Km = 10(4) M-1 s-1). The pH optimum for the cleavage of the native, the oxidized, the Met358-->Leu mutated inhibitor, and 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, a synthetic Pseudomonas elastase substrate are, 6.0, 7.0, 6.5 and 5.8, respectively. We conclude that P. aeruginosa elastase readily hydrolyzes substrates with P'1 methionine or alanine residues and that its pH optimum is not as alkaline as usually thought.

Amino Acid Sequence↗
PubMed1993

Bovine stefin C, a new member of the stefin family.

Four low M(r) cysteine proteinase inhibitors with different pI values were isolated from bovine thymus using alkaline activation of the gland homogenate, affinity chromatography on carboxymethyl-papain-Sepharose, gel filtration on Sephadex G-50, ion-exchange chromatography on a DEAE-cellulose column and a fast protein liquid chromatography Mono Q column, and hydrophobic chromatography on a TSK Phenyl-5 PW column. One of the inhibitors was identified both as the monomeric and dimeric forms of stefin B. Two others, called cysteine proteinase inhibitor-1 and cysteine proteinase inhibitor-2, were N terminally blocked and most likely belong to the stefin family. The complete amino acid sequence of the last inhibitor, namely bovine stefin C, was determined. The inhibitor consisted of 101 amino acids and its M(r) was calculated to be 11,546. It exhibits considerable sequence homology with other inhibitors from the stefin family. It was identified as the first tryptophane-containing stefin and it had a prolonged N terminus. The four inhibitors had similar inhibitory activities on cysteine proteinases. They were fast-acting inhibitors of papain and cathepsin L (kass > or = 1.8 x 10(6) M-1 s-1) and formed very tight complexes with the enzymes (Ki < or = 180 pM). In contrast, they were relatively poor inhibitors of cathepsin B (Ki > 100 nM).

Amino Acid Sequence↗
PubMed1993

Kinetics of the pH-induced inactivation of human cathepsin L.

Cathepsin L is known as the most unstable lysosomal cysteine proteinase at neutral or alkaline pH. The kinetics of inactivation of human cathepsin L was studied by mixing the enzyme with a substrate and recording the release of product. The inactivation was found to be a first-order process, and the rate of the process decreased with the substrate concentration. The substrate-independent inactivation rate constant kinact was found to be 0.15 s-1 at pH 7.4 and 37 degrees C and increased 85-fold between pH 7.0 and 8.0. At pH 7.4, kinact increased 3200-fold between 5 and 37 degrees C with an energy of activation 174.7 kJ/mol. Inactive cathepsin L did not reactivate at pH 5.5. The rate of inhibition of cathepsin L by stefin B or chicken cystatin at pH 7.4 was much faster than the rate of spontaneous inactivation of the enzyme. The stefin B-cathepsin L complex incubated at pH 7.4 released active enzyme at pH 5.5, suggesting that the cysteine proteinase inhibitors might act as extracellular carriers of the cysteine proteinases.

Cathepsin L↗
PubMed1992

Heparin-induced conformational change and activation of mucus proteinase inhibitor.

Low molecular mass heparin (5.1 kDa) forms a tight complex with mucus proteinase inhibitor, the physiologic neutrophil elastase inhibitor of the upper respiratory tract. This binding strongly enhances the intrinsic fluorescence of the inhibitor and the rate of neutrophil elastase inhibitor association. One mole of this heparin fragment binds 1 mol of inhibitor with a Kd of 50 nM. From the variation of Kd with ionic strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy i due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy is due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. and (iii), about one-third of low quantum yield of Trp30, the single tryptophan residue of the inhibitor, blue-shifts its maximum emission wavelength by 6 nm, decreases the acrylamide quenching rate constant by a factor of 4, and increases the mean intensity weighted lifetime by a factor of 2.5. These important spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment. Heparin accelerates the inhibition of elastase in a concentration-dependent manner. When both enzyme and inhibitor are saturated by the polymer, the second-order association rate constant is 7.7 x 10(7) M-1 s-1, a value that is 27-fold higher than that measured with the free partners. This finding may have important physiologic and therapeutic bearing.

Amino Acid Sequence↗
PubMed1992

The proteinase: mucus proteinase inhibitor binding stoichiometry.

In the nanomolar enzyme and inhibitor concentration range, 1 mol of mucus proteinase inhibitor (MPI) inhibits 1 mol of neutrophil elastase, cathepsin G, trypsin, and chymotrypsin. In the micromolar concentration range, the enzyme:inhibitor binding stoichiometry is still 1:1 for elastase but shifts to 2:1 for the three other proteinases. These data could be confirmed by three nonenzymatic methods: (i) fluorescence anisotropy measurements of mixtures of proteinases with 5-dimethylaminonaphthalene-1-sulfonylated or fluoresceinylated MPI, (ii) absorption spectrocospy of fluorescein-MPI-proteinase complexes isolated by gel filtration, (iii) analytical ultracentrifugation which showed that the molecular mass of the MPI-chymotrypsin complex is 56 kDa, whereas that of the MPI-elastase complex is 39 kDa. The binary MPI-elastase complex is unable to inhibit trypsin or cathepsin G. On the other hand, 1 mol of elastase displaces 2 mol of trypsin or cathepsin G from their ternary complexes with MPI.

Animals↗
PubMed1992

Oxidized alpha 1-proteinase inhibitor: a fast-acting inhibitor of human pancreatic elastase.

Unlike human neutrophil elastase or porcine and rat pancreatic elastases, human pancreatic elastase is rapidly inhibited by oxidized alpha 1-proteinase inhibitor. The second-order association-rate constant for the reaction of the oxidized inhibitor with this enzyme (kass = 10(5) M-1 s-1) is only 8-fold lower than that measured with native alpha 1-proteinase inhibitor. Elastase releases faster from its complex with the oxidized inhibitor (t1/2 approximately 0.7 days) than from its complex with the native inhibitor (t1/2 approximately 5 days). Oxidized alpha 1-proteinase inhibitor is as efficient as the native inhibitor in inhibiting the elastolytic activity of elastase. Oxidized alpha 1-proteinase inhibitor may thus be considered as a physiological inhibitor of human pancreatic elastase which may prevent degradation of blood vessel elastin during acute hemorrhagic pancreatis.

Humans↗
PubMed1992

Heparin interferes with the inhibition of neutrophil elastase by its physiological inhibitors.

Heparin depresses the second-order rate constant kass for the inhibition of neutrophil elastase by alpha 1-proteinase inhibitor. For high and low molecular weight heparin the decrease in kass is 290-fold and 40-fold, respectively. This is due to a tight binding of the polymer to elastase: Kd = 3.3 nM or 89 nM for high or low molecular weight heparin respectively. In contrast heparin increases the rate of inhibition of elastase by mucus proteinase inhibitor. For low molecular weight heparin, there is a 27-fold increase in kass. This is due to a strong binding of the polymer to the inhibitor (Kd = 50 nM) which undergoes a conformational change.

Chromatography, Affinity↗
PubMed1992

Secretory leucocyte proteinase inhibitor: inhibition of fibronectin degradation by neutrophil elastase.

Degradation of surface-bound fibronectin of the upper respiratory tract by human leucocyte elastase (HLE) was shown to favour colonization of these mucosal surfaces by Gram-negative bacteria. We investigated the degradation of fibronectin by purified HLE and by enzymes released from stimulated human polymorphonuclear leucocytes (PMNs), in the presence of recombinant secretory leucocyte proteinase inhibitor (rSLPI) and alpha 1-proteinase inhibitor (alpha 1-PI), the two main inhibitors of HLE within the airways. Our results show that HLE degraded fibronectin at concentrations as low as 0.2 nM. To inhibit the degradation of fibronectin by pure HLE in an experimental system in which the enzyme was premixed with inhibitor, a twofold molar excess of rSLPI and an equimolar concentration of alpha 1-PI were required. On the other hand, a fivefold molar excess of rSLPI was necessary to inhibit degradation of fibronectin by enzymes released from stimulated neutrophils. In order to estimate the role of oxidants generated by stimulated PMNs in the activation of the inhibitory capacity of rSLPI by stimulated PMNs, we preincubated PMNs with antioxidants such as superoxide dismutase, methionine, catalase or Na-azide prior to stimulation of the cells. Under these conditions, a threefold molar excess of rSLPI over released HLE was required to inhibit the degradation of fibronectin, raising the possibility that either exogenous or endogenous antioxidants in the lung could be important in improving the efficacy of this therapeutic antiprotease. We conclude that a molar excess of rSLPI to HLE is always necessary to inhibit fibronectin degradation by HLE, and that addition of antioxidants partly prevents the inactivation of rSLPI by oxidants released from stimulated PMNs.

Antioxidants↗
PubMed1991

Kinetics of the interaction of chymotrypsin with eglin c.

The kinetics of binding of recombinant eglin c to bovine pancreatic chymotrypsin was studied by conventional and stopped-flow techniques. With nanomolar enzyme and inhibitor concentrations, the inhibition was fast and pseudo-irreversible (k(assoc.) = 4 x 10(6) m-1.s-1 at 7.4 and 25 degrees C). Reaction of the enzyme-inhibitor complex with alpha 1-proteinase inhibitor, an irreversible chymotrypsin ligand, resulted in a slow release of free eglin c, which was monitored by electrophoresis (k(dissoc.) approximately 1.6 x 10(-6) s-1, t1/2 approximately 5 days). The proflavin displacement method and a stopped-flow apparatus were used to monitor the association of chymotrypsin with eglin c under a wide range of inhibitor concentration and under pseudo-first-order conditions. At pH 7.4 and 25 degrees C or 5 degrees C, or at pH 5.0 and 25 degrees C, the pseudo-first-order rate constant of proflavin displacement increased linearly with eglin c up to the highest concentration tested, suggesting a one-step bimolecular association reaction: E + I in equilibrium with EI. However, kassoc. is much lower than the rate constant for a bimolecular reaction and its activation energy (66 kJ.mol-1 at pH 7.4 and 78 kJ.mol-1 at pH 5.0) is far too high for a diffusion-controlled step. The enzyme-inhibitor association may therefore occur via a loose pre-equilibrium complex EI* (Ki* much greater than 5 x 10(-4) M) that rapidly isomerizes (k2 much greater than 2 x 10(3) s-1) into an extremely stable final complex (Ki approximately 4 x 10(-13) M). Unlike other proteinase-inhibitor systems, the chymotrypsin-eglin association is virtually pH-independent.

Amino Acid Sequence↗