Human alpha 2-macroglobulin as a cytokine-binding plasma protein. A study with rh-interleukin-1 beta and rh-interleukin-6.
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Chymotrypsin↗
Biomedical subjects
E Delain
Publications and source records attributed to E Delain.
Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase.
The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees).
Base Sequence↗
Electron microscopy of macromolecules. The French contribution.
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Animals↗
Electron microscopy of the nucleocapsid from disrupted Moloney murine leukemia virus and of associated type VI collagen-like filaments.
To analyze the constituents of retroviruses, the Moloney murine leukemia virus was disrupted and observed by dark-field electron microscopy. Virus disruption was achieved by several methods: osmotic shock, freezing-thawing cycles, and exposure to urea up to 4 M, to NaCl up to 1 M, and to Triton X-100. Several components associated with broken Moloney murine leukemia virus were repeatedly found in preparations. These components have been described as rings, thick filaments, chain-like filaments, threads covered with proteins, threads with buckles, and naked threads. A quantitative analysis of the occurrence of these components has been carried out. Among them, the thick filaments composed of a compact helical arrangement of small beads 5 nm in diameter were considered to represent the nucleocapsid. The protease-sensitive buckles found on some threads could be a compact form of the viral RNA associated to the nucleocapsid protein NCp10. The RNase-sensitive naked threads are interpreted as the deproteinized viral RNA itself. The ubiquitous chain-like filaments possess a periodic structure identical to that of polymerized type VI collagen. It is proposed that this adhesive protein is associated with the viral envelope taken from the cell membrane during the budding process of retroviruses.
Capsid↗
Ultrastructure of alpha 2-macroglobulins.
New results concerning the ultrastructure of human alpha 2-macroglobulin (alpha 2M) molecules are presented in connection and comparison with the historical, the current and our own most recent, even unpublished results on the structure and function of alpha 2M and related proteins. The electron microscopic approach uses classical negative staining, combined with the new imaging mode "Electron Energy Loss Spectroscopy", which provides unusual contrast, resolution and readability of the electron micrographs. Immuno- and cryoelectron microscopy, as well as image processing has provided new data necessary to the building of tentative 3D models of the molecule. A model for the native tetrameric alpha 2M is described for the first time, and tries to explain and gather the various observations, sometimes contradictory, taken from different laboratories. A revised version for a model of the methylamine- and proteinase-transformed forms of alpha 2M is also shown. The probable positions of the bait regions and the thiol esters are given on both models. We confirm that alpha 2M is a twin trap capable of inactivating one or two proteinases by partial immobilization. Preliminary results on the production of crystals of alpha 2M-chymotrypsin complexes are also presented. A critical analysis of our models is presented in comparison with others. The technical limitations reached with some techniques and some possible extensions of future research in the field are also presented.
Animals↗
Electron microscopic observations of the effects of anthraquinone derivatives on plasmid DNA.
Electron microscopy was used to analyse the precipitation of DNA observed when mixed with two tripeptide derivatives of mitoxantrone, with or without a 5,8-dihydroxy group (DHQ-GHK and Q-GHK, respectively) on the anthraquinonic ring. This precipitation was compared to that obtained with the basic drugs, mitoxantrone (DHAQ) and ametantrone (AQ). The effects of these compounds on the supercoiling of form I and the lengthening of form II of pBR322 DNA molecules, respectively, were evaluated. A strong lengthening of the DNA molecules was observed for ametantrone (max: 57%), but only 32% for Q-GHK, both at r (drug/base pari) = 250. With the dihydroxy derivative DHQ-GHK, it was not possible to show more than a 10% increase in length because DNA molecules were not measurable at r greater than 100. Only Q-GHK relaxed supercoiled molecules at the low r values of 10. Complex phenomena of condensation-precipitation were observed with these two tripeptide derivatives. In addition to a strong lengthening of form II DNA molecules, AQ induced specifically the formation of toruses, and DHAQ that of large organized DNA condensation. The variety of the aggregations is described and discussed with regard to the antitumor properties of these derivatives, and the literature concerning the various descriptions of DNA aggregation.
Anthraquinones↗
Quantitative electron microscopic analysis of DNA-protein interactions.
Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.
Animals↗
Localization of the proteinases in the human alpha 2-macroglobulin-chymotrypsin complex by image processing of electron micrographs.
Human alpha 2-macroglobulin (alpha 2M), a large tetrameric plasma glycoprotein, inhibits a wide spectrum of proteinases by a particular "trapping" mechanism resulting from the proteolysis of peptide bonds at specific "bait" regions. This induces the hydrolysis of four thiol esters triggering both the possible covalent bonding of the proteinases and a considerable structural change in the alpha 2M molecule, also observed following direct cleavage of the thiol esters by methylamine. By subtracting average images of electron micrographs from two populations of alpha 2M molecules in the same biochemical state (with both the four cleaved bait regions and thiol esters), but containing either two or zero chymotrypsins, we are able to demonstrate the position of the two proteinases inside the tetrameric alpha 2M molecule. The comparison of the alpha 2M molecules transformed either by immobilized chymotrypsin or methylamine shows that the proteolysis of the bait regions seems of minimal importance for the general shape of the molecule and provides a direct visualization of the actual role of the thiol esters in the conformational change.
Amino Acid Sequence↗
Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.
The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Image processing of proteinase- and methylamine-transformed human alpha 2-macroglobulin. Localization of the proteinases.
Comparative x-ray scattering experiments and electron microscopic observations have been performed on native S-form, and on different F-forms of human plasma alpha 2-macroglobulin (alpha 2M), obtained by proteinase (chymotrypsin, plasmin, and thrombin) or methylamine treatment. Image processing of electron micrographs of the alpha 2M molecules transformed by chymotrypsin, plasmin, and methylamine displayed average images which could be compared. The proteinase-complex alpha 2M molecules exhibited the usual H-like structure, but the methylamine-inactivated ones showed a different organization, with almost no stain-excluding material in the central region of the molecule, which therefore presented a central cavity filled with stain. By subtracting average images of alpha 2M-methylamine from alpha 2M-chymotrypsin or alpha 2M-plasmin, a putative localization of the proteinases inside the alpha 2M molecule, very close to its center was revealed. The values of the radii of gyration for the S- and F-forms obtained by x-ray scattering were very different (78 and 67.7 A, respectively). All four scattering curves of the F-forms were comparable in shape and showed maxima and minima different from that of the S-form alpha 2M. Image processing of electron micrographs and x-ray scattering have provided independent results which indicate that a large cavity exists in the alpha 2M-methylamine molecule and that the proteinases might be located in a very central position inside the alpha 2M-proteinase molecules.
Chymotrypsin↗
Dissociation of alpha 2 M-macroglobulin into functional half-molecules by mild acid treatment.
A slight decrease in pH below neutrality causes the dissociation of alpha 2-macroglobulin (alpha 2M) into dimers formed of two disulfide-bonded subunits. Half-dissociation occurs at pH 6.30 (50 mM NaCl), as determined by gel filtration analysis. The dissociation can be reversed either by increasing the pH or the ionic strength. The ability of alpha 2 M half-molecules at pH 5.75 to bind chymotrypsin is not too different from that of the whole molecule at pH 7.5. Furthermore, the steady-state kinetic parameters toward chromogenic substrate of chymotrypsin bound to alpha 2 M half and whole molecules are quite identical. Likewise, the accessibility of trypsin toward soybean trypsin inhibitor is also fairly similar when involved in half or whole alpha 2 M complexes. These results are consistent with the idea that alpha 2 M-half molecules on chymotrypsin binding undergo a conformational change. This change can be observed by electron microscopy.
Chromatography, Gel↗
The Xenopus laevis mitochondrial protein mtDBP-C cooperatively folds the DNA in vitro.
The binding of the Xenopus laevis mitochondrial protein mtDBP-C to DNA was studied by equilibrium density banding, agarose gel electrophoresis and electron microscopy. The results obtained show that the mtDBP-C binds cooperatively to DNA irrespective of whether the DNA is supercoiled, relaxed or linear and it induces the formation of superhelical turns locally leading to the formation of a highly folded structure. It appears that this protein could be involved in the compaction of DNA in the mitochondrial nucleoid.
Animals↗
Cytoplasmic accumulation of ditercalinium in rat hepatocytes and induction of mitochondrial damage.
Ditercalinium (NSC 335153), a 7H-pyridocarbazole dimer, is a bis-intercalating agent whose mechanism of action differs from that of other mono-intercalating compounds, such as ellipticine derivatives. After a Phase I clinical trial, where irreversible hepatotoxicity was the dose-limiting side-effect, we have reinvestigated the disposition of ditercalinium in rats after i.v. administration. Tissue distribution of the tritiumlabeled drug was studied during 5 weeks. The drug distributed very quickly into tissues, and accumulated mostly in liver and kidneys. A much slower clearance followed, with a half-life greater than 7 days. The present study raises the question of whether ditercalinium, a biscationic lipophilic agent, exerts a direct mitochondria interaction both in vitro and in vivo. Fluorescence microscopy on rat tissue cryosections, after drug administration, showed that the major cellular site of drug accumulation corresponds to mitochondria. The mitochondrial probe 3,3'-diethyloxadicarbocyanine confirmed the mitochondrial localization of ditercalinium. An identical fluorescent pattern was found in cultured rat hepatocytes. These fluorescent granulation patterns suggest mitochondrial damage. To further study cell alterations, ultrastructural changes in rat liver and kidneys were observed with selective mitochondrial damage while nuclei remained apparently normal. The same observations were made in rat hepatocyte cultures. Therefore, the accumulation of ditercalinium in tissues and, more particularly, in mitochondrial probably plays an important role in ditercalinium-induced toxicity.
Animals↗
The molecular organization of human alpha 2-macroglobulin. An immunoelectron microscopic study with monoclonal antibodies.
To study the three-dimensional organization of alpha 2-macroglobulin (alpha 2M) from human plasma, immunoelectron microscopy of negatively stained specimens was used. A panel of monoclonal antibodies (mAb) with specificities typical for the two major conformers of alpha 2M (native and protease-transformed) was explored. The mAb have been selected and were classified biochemically as specific for either native or transformed alpha 2M or as reactive with both conformers. Furthermore, among the mAb that were specific for the proteinase-transformed form of alpha 2M, those reacting with the 20-kDa receptor-binding domain were considered a fourth category. Immunoelectron microscopy with these 20-kDa receptor-binding domain-specific mAb yielded the most typical result: predominantly, individual H-like alpha 2M-chymotrypsin molecules were complexed with two IgG molecules, each one bound to the extremities of two arms of the H-like figure. The resulting planar complex has the appearance of a dumbbell. Since this was observed with eight different mAb of this specificity, the result is interpreted to mean that the 20-kDa receptor-binding domain is compact and constitutes the outermost domain at the extremes of the arms of the H-like transformed alpha 2M. The mAb which are specific for the transformed state of alpha 2M but which do not react with the 20-kDa receptor-binding domain, also bound at the arms of the H-like figure, but at nonterminal positions. Moreover, these mAb produced mostly linear, chain-like immune complexes of numerous H-like alpha 2M molecules cross-linked by the IgG. The large category of mAb that reacted with both conformers of alpha 2M (native and proteinase complex) were observed to make various types of immune complexes with intra- and intermolecular cross-linking by the IgG. The observations of reaction of these mAb with Cd2+-induced dimers (half-molecules of alpha 2M), either native or transformed, proved helpful and, for certain mAb, essential to understand the organization of the alpha 2M-IgG complexes. Combined, the observations allow us to propose new models for the three-dimensional organization of native and chymotrypsin-transformed dimeric and tetrameric human alpha 2M.
Antibodies, Monoclonal↗
Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+.
Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.
Cadmium↗
Image analysis and three-dimensional model of chymotrypsin-transformed human alpha 2-macroglobulin complexed with a monoclonal antibody specific for this conformation.
Alpha 2-macroglobulin (alpha 2M) is a plasma inhibitor of proteinases, the steric mechanism of which is based on a considerable conformational change. The typical and distinct H-like shape of alpha 2M-chymotrypsin (alpha 2M-chy) complexes seen by electron microscopy led us to an ultrastructural study of the binding of a monoclonal antibody (Mab) specific for this conformation of alpha 2M. The epitope of this Mab is located near the extremities of the 4 arms of the H-like alpha 2M-chy, at a site that is not accessible on the native molecule. The identical binding of the Mab on the 4 arms of the tetrameric molecule demonstrates that these arms are equivalent portions of the 4 monomers. Various types of immune complexes between alpha 2M and IgG are described, and images of individual immune complexes were processed by correspondence analysis. This extracts new information concerning the organization of chymotrypsin-transformed alpha 2M. The molecule appears asymmetrical, presents 2 conformational states (which we describe as relaxed and twisted), and has flexible arms. These intramolecular motions are supposed to be related to IgG binding. The results are discussed in comparison with previously published models of proteinase-transformed alpha 2M.
Antibodies, Monoclonal↗
Functional alpha 2-macroglobulin half-molecules induced by cadmium.
Human alpha 2-macroglobulin can be reversibly dissociated by Cd2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole alpha 2M molecules. Cd2+ can also induce the dissociation of plasmin- and chymotrypsin - alpha 2M complexes into proteinase-alpha 2M half-molecule conjugates. These results, taken with the fact that monomeric units of alpha 2M cannot bind these proteinases, strongly suggest that each active site of alpha 2M consists in a specific arrangement of two monomeric units linked by disulfide bridges.
Binding Sites↗
The covalent and non-covalent binding modes of elastase with alpha 2-macroglobulin influence the conformation of the protease.
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Binding, Competitive↗