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Biomedical subjects

F Pochon

Publications and source records attributed to F Pochon.

At least 37 records · Page 2Linked to original sources
PubMed1986

Specific fluorescent labeling of alpha 2-macroglobulin-bound proteases: accessibility and localization of their active site within the complex.

Pyrenebutylmethylphosphonofluoridate reacts with trypsin and elastase to yield a conjugate with a stoichiometry of one fluorescent label per enzyme molecule as already observed with chymotrypsin. The kinetics of inactivation indicate that the serine active center of the proteases is involved in the labeling reaction. The binding of the proteases to alpha 2-macroglobulin does not modify the specificity of the reaction but drastically diminishes the labeling rate which also depends upon alpha 2-macroglobulin protease binding ratio. Dynamic quenching of the conjugated pyrene moiety by acrylamide, and iodide ions is markedly reduced upon reaction of the protease with alpha 2-macroglobulin, indicating a reduced accessibility of the protease active center in the complex. Singlet--singlet energy transfer measurements from the donor pyrene labeled active center of the proteases to the alpha 2-macroglobulin acceptor labeled thiol groups which are liberated upon protease fixation, gave a rough estimate of the distance (about 25 A) between the active center of the two alpha 2-macroglobulin bound protease molecules.

Binding Sites↗
PubMed1986

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations.

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.

Cations, Divalent↗
PubMed1984

Interaction of alpha 2-macroglobulin-bound thrombin with hirudin.

The human thrombin bound to alpha 2-macroglobulin (alpha 2 M) in a 1:1 stoichiometry is still able to interact with one of its specific inhibitors, hirudin. The dissociation constant of the complex hirudin--alpha 2M-bound thrombin is 1 X 10(-7) M, whatever the mode of thrombin binding, covalent or non-covalent.

Chromatography, Gel↗
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

Evidence for rotational contribution to protein-facilitated proton transport.

Two modes of molecular motion of carrier molecules can, in principle, lead to a facilitated transport of a substrate: translational and rotational diffusion. In the present study, which deals with the mechanism of the facilitated diffusion of H+ and O2 in solutions of earthworm hemoglobin, examples for both types of facilitation are presented. Only translational, not rotational, diffusion of earthworm hemoglobin appears to lead to a facilitated O2 flux. In contrast, substantial facilitated H+ fluxes of comparable size arise from rotational diffusion as well as from translational diffusion of this large protein. This is derived from measurements of facilitated H+ and O2 fluxes in earthworm hemoglobin solutions and determinations of the rotational and translational diffusion coefficients of earthworm hemoglobin with the help of a theoretical treatment of facilitated diffusion by rotational carrier diffusion. H+ transport by rotational protein diffusion appears to be a case where the often-postulated mechanism of facilitated transport by rotation of a carrier lends itself to experimental verification.

Biological Transport↗
PubMed1983

The alpha 2 macroglobulin/thrombin interaction in the presence of hydroxylamine.

The influence of a primary amine, hydroxylamine, on the interaction between alpha 2 macroglobulin (alpha 2M) and thrombin was analyzed by electrophoretic and enzymatic methods. Hydroxylamine (final concentrations 0.01 M and 0.1 M) was added to the alpha 2M solution 3 to 5 min before thrombin. In these conditions hydroxylamine had no direct influence on alpha 2M itself. The inhibition of thrombin activity by alpha 2M was still possible and alpha 2M/thrombin complexes were observed. However the rate of inhibition of the clotting activity of thrombin was diminished in function of the hydroxylamine concentration. The complexes obtained in the absence of the nucleophylic agent were resistant to SDS dissociation, whereas those obtained in the presence of hydroxylamine were dissociated by SDS. In both cases, the amount of alpha 2M polypeptide chains cleaved by thrombin was the same (50%). In conclusion, hydroxylamine does not prevent the formation of alpha 2M/thrombin complexes, but it reduces the covalent binding of the enzyme to the inhibitor in a concentration dependent fashion, leading to the formation of "abnormal" complexes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c.

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.

Ascomycota↗
PubMed1983

Structural arrangement in the alpha 2-macroglobulin--thrombin complex.

The cysteine sulfhydryl groups of alpha 2-macroglobulin (alpha 2M) generated upon thrombin complex formation are in contact with the proteinase surface as evidenced by singlet--singlet energy transfer measurements from N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid-labeled thiol functions of alpha 2M to fluorescein isothiocyanate-labeled thrombin. The thrombin-alpha 2M binding is normally covalent, but the presence of hydroxylamine during the reaction leads to the formation of a non-covalent complex. The transfer energy determinations show that the alpha 2M binding sites of thrombin are quite similar, whatever covalent or non-covalent binding occurs.

Chymotrypsin↗
PubMed1983

Localization of the proteinase-induced thiol groups in alpha 2-macroglobulin.

Free thiol groups released on proteolytic attack of alpha 2-macroglobulin by trypsin or chymotrypsin bind covalently to thiopropyl-Sepharose, indicating that they are located at the surface of the complexes. These cysteine sulfhydryl groups appear to be in contact with the alpha 2M-bound proteases from singlet-singlet energy transfer measurements between fluorescein isothiocyanate-labeled proteinases and N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid-labeled thiols in alpha 2-macroglobulin.

Binding Sites↗
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

Heparin and the progressive anti-thrombin activity of alpha 2-macroglobulin.

Interactions between alpha 2-macroglobulin (alpha 2M) and thrombin have been studied by spectroscopic, isotopic and electrophoretic methods in presence or in absence of heparin. It is shown that thrombin binds to alpha 2M in a 1:1 ratio. Fluorescamin labelled heparin of Mr 7 000 interacts with thrombin to form a 2:1 molar complex. This complex does not bind to alpha 2M and is unable to achieve any proteolytic cleavage of this protein. In contrast the interaction of alpha 2M with chymotrypsin is not significantly affected by the mucopolysaccharide. Moreover, heparin is unable to react with alpha 2M bound thrombin.

Antithrombins↗
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↗
PubMed1981

Localization of the two protease binding sites in human alpha 2-macroglobulin.

The distance between the two protease binding sites in human plasma alpha 2-macroglobulin has been estimated using singlet-singlet energy transfer experiments. alpha-Chymotrypsin was labeled covalently with donor (dansyl chloride) or acceptor (fluorescein isothiocyanate) groups, and the efficiency of transfer between these dyes was measured within the alpha 2-macroglobulin . (alpha-chymotrypsin)2 complex. The distance between the surface exterior of the protease molecules was calculated to be 4 to 11 A, depending on the assumption made about the equivalence of the binding sites. A catalytically active dimer of alpha-chymotrypsin was prepared using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. In contrast with the alpha-chymotrypsin monomer, it binds to alpha 2-macroglobulin with a 1:1 stoichiometry. However, the 1:1 alpha 2-macroglobulin . dimeric alpha-chymotrypsin complex is still able to bind 1 mol of the alpha-chymotrypsin monomer. Energy transfer experiments performed with this ternary complex showed that the distance between the alpha 2-macroglobulin-bound alpha-chymotrypsin molecules is not higher than 4 A, i.e. the two protease binding sites in alpha 2-macroglobulin should be about 44 A apart (center to center) if the anhydrous radius of alpha-chymotrypsin is 20 A.

Animals↗
PubMed1981

The interaction of the membrane binding segment of cytochrome b5 with phospholipid vesicles. A pulse-radiolysis investigation.

The reactions of the radical anions Br2- and (SCN)2- produced by pulse-radiolysis have been used to study the interaction of dipalmitoyl phosphatidyl choline vesicles with the membrane binding segment (MBS) of cytochrome b5. Tryptophan oxidation by Br2- at neutral pH was characterized spectrophotometrically in detergent-solubilized solutions of MBS; the attack of tyrosine by (SCN)2- under alkaline conditions could also be observed directly. The incorporation of MBS into the lipid bilayer protected the tryptophan, tyrosine and methionine residues from oxidation by Br2- or (SCN)2-. Some structural implications of these results are discussed.

Cytochromes↗
PubMed1980

Structure-activity relationships in a series of newly synthesized 1-amino-substituted ellipticine derivatives.

The synthesis of a series of 1-amino-substituted pyrido[4,3-b]carbazole derivatives, based on the substitution of corresponding 1-chloroellipticines, is reported. The cytotoxic properties on tumor cells grown in vitro, the in vivo acute toxicity of the most potent in vitro cytotoxic compounds, and the antitumor properties toward the L1210 leukemia system are described. No correlation between the apparent association constant to DNA and the in vitro cytotoxicity or the in vito antitumor efficiency could be observed in this series. 9-Hydroxylated derivatives were more cytotoxic in vitro than the corresponding 9-methoxylated compounds. However, their antitumor efficiencies on the in vivo experimental systems do not confirm the advantage of demethylation. The presence of a [(dialkylamino)alkyl]amino side chain at the 1 position of ellipticines increases the antitumor potency: 1-[[3-(diethylamino)propyl]amino]-5,11-dimethyl-6H-pyrido[4,3-b]carbazole (5) is a very potent antitumor compound (% ILS of 134 on the L1210 leukemia system).

Alkaloids↗