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Biomedical subjects

M Dautrevaux

Publications and source records attributed to M Dautrevaux.

At least 37 records · Page 2Linked to original sources
PubMed1987

Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation.

Rat-liver glucocorticoid receptor was incubated with either [3H]triamcinolone acetonide or [3H]RU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following pI values: 5.3 +/- 0.2 (n = 17) and 4.4 +/- 0.1 (n = 17). All these peaks correspond with receptor isoforms as suggested by control experiments. The receptor state was analyzed after focusing by a chromatographic assay on DNA-cellulose, DEAE-trisacryl and hydroxyapatite minicolumns. The peak of pI 4.4 apparently corresponded to the non-transformed receptor and was greatly stabilized in the presence of RU 486, whereas the peaks of pI 4.8 and 5.3 were probably made of transformed receptor and meroreceptor. These results were confirmed by autoradiographic studies after isoelectric focusing of receptor molecules covalently labelled with [3H]dexamethasone mesylate. Thus, the rat-liver glucocorticoid receptor appeared to be a rather acidic protein which became less acidic after transformation by heat, displaying a pI shift which was strongly reduced in case of steroid-receptor complexes formed with the antiglucocorticoid RU 486.

Animals↗
PubMed1987

[Preparation of an anti-idiotypic antibody directed against anti-dexamethasone antibody and demonstration of its ability to acknowledge the receptor of glucocorticoids].

By using a 3-carboxymethyloxime dexamethasone derivative coupled to bovine serum albumin we have prepared specific anti-dexamethasone antibodies in rabbits. These antibodies were then purified by affinity chromatography and administered to a second set of rabbits. One of them produced anti-idiotypic antibodies able to impede the [3H] dexamethasone binding of the initial anti-dexamethasone antibodies and to displace the [3H] dexamethasone-antibodies complexes towards high molecular weight species in gel filtration experiments. Moreover these antibodies we were also able to impede the binding of [3H] dexamethasone to the rat liver glucocorticoid receptor and to recognize the highly purified receptor using Western blot analysis.

Animals↗
PubMed1986

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography.

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.

Antibodies↗
PubMed1986

Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU 486.

We have compared the physicochemical characteristics of the non-activated (molybdate stabilized) glucocorticoid-receptor complex bound either to [3H]triamcinolone acetonide or to the antagonist [3H]RU 486 with those of the activated (25 degrees C preheated) complexes. The level of activation was measured by a DNA cellulose assay. The physicochemical features of the steroid-receptor complexes were analyzed by various techniques including high-performance size exclusion chromatography, sucrose gradient sedimentation and high-performance DEAE ion exchange chromatography. All the buffers used during the analytical procedures contained sodium molybdate in order to prevent any dissociation of the steroid-receptor complex. The non-activated glucocorticoid receptor bound either to [3H]TA or to [3H]RU 486 sedimented at 9.3S on sucrose gradient, displayed at 70 A Stokes radius in high performance size exclusion chromatography on TSK G4000 SW column, and was eluted at 0.22-0.28 M KCl by anion-exchange chromatography on a DEAE 545 column. After activation the Stokes radius and the sedimentation coefficient declined to 50 A and 4.5S respectively, and the complex was eluted by 0.10-0.12 M KCl on the ion-exchange column. No qualitative difference could be detected between the characteristics of the glucocorticoid-receptor complexes bound either to [3H]TA or to [3H]RU 486. Moreover, the relative distribution of "non-activated" and "activated" forms of the glucocorticoid receptor obtained through physicochemical experiments was highly correlated with the activation level determined by DNA binding experiments. However the activation level of [3H]RU 486-glucocorticoid-receptor complex appeared markedly decreased (15%) when compared with that of the agonist [3H]TA-receptor complex (65%). Experiments done with an antagonist steroid of the 17 beta-carboxamide series of dexamethasone exhibit the same results with an activation level of 12% as compared with triamcinolone acetonide. Thus, two antihormones which have different structures show the same behavior towards the glucocorticoid-receptor complex by strikingly reducing both the activation process and the size reduction of the steroid-receptor complex which is concomitant to activation and which could constitute the first step of this process.

Animals↗
PubMed1985

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.

Animals↗
PubMed1985

Covalent chromatography by thiol-disulfide interchange of the highly-purified non-transformed rat liver glucocorticoid-receptor.

Highly-purified non-transformed rat liver [3H]triamcinolone acetonide-receptor complex was shown to be covalently adsorbed on activated thiol sepharose 4B, a reactive sulfhydryl matrice. Elution by mercaptoethanol in excess and inhibition of binding by previous treatment of the complex with N-ethylmaleimide clearly demonstrated the specificity of the binding by thiol disulfide interchange. The transformed [3H]triamcinolone acetonide-receptor complex, partially purified by DNA-cellulose chromatography, was also retained on activated thiol sepharose 4B. The physicochemical characteristics of both the transformed and non-transformed glucocorticoid receptor complexes eluted from the covalent chromatography column were studied by HP size exclusion chromatography on a TSK G 3000 SW column and were found to be identical to those of the starting complexes. These results provide direct evidence for accessible sulfhydryl groups on the glucocorticoid receptor complex surface, probably distinct from the steroid binding essential sulfhydryl group.

Animals↗
PubMed1984

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures.

A series of N-alkylmaleimides was shown to inactivate effectively the rat liver glucocorticoid receptor at neutral pH. A partial purification of the unbound cytosolic receptor by protamine sulfate precipitation and a careful stabilization of the essential thiol by dithiothreitol and sodium molybdate before the alkylation step appeared essential to obtain pseudo-first-order kinetics. Moreover, performing the experiment at -12 degrees C in buffer containing 40% glycerol as antifreeze agent resulted in increased receptor stabilization and a slowing-down of the inactivation process, which could then be more accurately studied. This process was demonstrated to be dose- and pH-dependent in the case of N-ethyl- and N-nonylmaleimides. Furthermore, comparison of the various N-alkylmaleimides revealed a striking increase of receptor inactivation with increasing chain length of the maleimide derivative. Full protection against inactivation was afforded by previous [3H]dexamethasone binding on the receptor. Long-chain N-alkylmaleimides inactivated by beta-mercaptoethanol were still able to inhibit the [3H]-dexamethasone binding noncovalently. Likewise N-nonylsuccinimide was shown to compete with [3H]dexamethasone for receptor binding. It is suggested that the chain length effect observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the receptor prior to the irreversible alkylation of sulfhydryl groups. These groups lie in a hydrophobic environment, probably in the steroid binding site itself.

Animals↗
PubMed1984

Special problems encountered during the cyanogen bromide cleavage of lobster arginine kinase.

During structural analysis of Lobster muscle arginine-kinase, we have isolated a CNBr resulting peptide with a blocked N-terminal residue. This peptide was sequenced after unblocking by mild acid treatment (1 N HCl at 100 degrees C for 10 min). The blocked form is not due to the formation of pyroglutamic acid nor is it due to the formation of diketopiperazine. We have applied the experimental conditions used for CNBr cleavage of lobster arginine-kinase to a synthetic peptide the structure of which is similar to the above CNBr peptide. We bring evidence that during CNBr cleavage partial formylation occurs with a possible cyclization of a 7 membered ring of Gly--Asp...

Amino Acid Sequence↗
PubMed1984

Physico-chemical properties and evidence for electrophoretic variants of rat transcortin.

Isolation of rat plasma transcortin was carried out by affinity chromatography, as previously described for human. The protein was shown to be pure by PAGE and one single N-terminal amino acid was identified (Ser), which suggested that the protein molecule has a single polypeptide chain. This assumption is supported by SDS-PAGE. The amino acid composition was reported and compared with the one of human transcortin. The purified protein always migrated in PAGE (with or without SDS) as a double band; the faster component being more intense than the slower one. Whether transcortin was free or bound to corticosterone, the same aspect was observed. Molecular weight of these two variants were determined by SDS-PAGE as 65,900 and 75,800. Polymers only appeared after irreversible denaturation of the protein, as previously described for human transcortin. Various other physical parameters were determined: a sedimentation coefficient of 3.71 S +/- 0.18 was calculated by ultracentrifugation in sucrose gradient, association constants at 4 degrees C for corticosterone and cortisol (2.7 X 10(9) M-1 and 4.2 X 10(8) M-1, respectively).

Amino Acids↗
PubMed1983

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate.

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.

Chloromercuribenzoates↗
PubMed1983

Importance of arginine residues in the determination of the biological activity of human corticosteroid-binding globulin.

The binding activity of human corticosteroid-binding globulin (CBG) is pH dependent and governed in alkaline pH ranges by the pK of arginine. No essential arginine residues is located in the binding site. The loss of biological activity is rapid and complete as soon as one arginine residue is modified by phenylglyoxal. There is also a transconformation of the CBG molecule. Therefore the surprising stability of CBG up to pH 11.5 may be explained by a large dependence of the CBG tertiary structure on the integrity of one arginine residue : as long as the ionized state of this single residue is not changed (pH less than pK of the guanidyl group) the tertiary structure of the biologically active CBG is maintained in alkaline pH ranges.

Arginine↗
PubMed1982

Studies of an abnormal serum albumin unstable upon storage.

A case of inherited bisalbuminemia was discovered in which electrophoretic controls showed that the proportion of the abnormal albumin decreased progressively during storage of the serum either at 4 degrees C or at room temperature. Such a decrease was also found when the serum was incubated with the proteolytic enzymes trypsin or plasmin. Studies with the isolated abnormal albumin showed that either during storage or after incubation with trypsin (or plasmin), its mobility became identical to that of normal serum albumin. Structural analyses showed that the albumin variant was identical to the previously described pro-albumin Christchurch that contains an additional N-terminal sequence: Arg-Gly-Val-Phe-Arg-Gln. It was therefore suggested that the progressive decrease of the abnormal albumin during storage by serum was related to the cleavage of the N-terminal abnormal sequence by plasmin already present in the serum. The decrease of the abnormal albumin during storage was inhibited by addition of cortisol to serum (1 mol per mol serum albumin). This was found related to a protective effect of cortisol bound to the albumin variant on proteolysis by plasmin.

Adolescent↗
PubMed1982

[Continuous haemofiltration in encephalopathy associated with hepatic failure (author's transl)].

Thirty-one comatose patients (18 with cirrhosis of the liver, 13 with severe hepatitis) were treated with continuous haemofiltration on polyacrylonitrile membrane (AN 69). The mean duration of sessions was 45 +/- 37 h, during which 136 +/- 108 1 of ultrafiltrate were dialyzed. Sixteen patients emerged from coma, 14 (7 with cirrhosis, 7 with hepatitis) completely and 2 partially. Blood ammonium levels decreased by 47 +/- 21% during the first 24 hours. Aminoacid clearance ranged from 20 to 50 ml/min, but only non significant changes were observed in the branched-chain/aromatic aminoacid ratio. Provided the haemodynamic balance is preserved, the technique is well tolerated. The duration of dialysis depends upon the degree of thrombopenia induced. Since the long-term prognosis of both cirrhosis patients and severe hepatitis patients is unmodified, continuous haemofiltration can only be helpful in cases where hepatic regeneration is possible; failing this, liver transplantation should be considered.

Amino Acids↗
PubMed1982

Modification of human transcortin by tetranitromethane. Evidence for the implication of a tyrosine residue in cortisol binding.

The effect of tetranitromethane on the cortisol binding activity of human transcortin has been investigated. This reagent induced a decrease of activity concomitant with nitration of tyrosine residues. An oxidation of sulphydryl groups was also observed but had no implication on cortisol binding. The nitration was specifically oriented in the site at pH6 and with low concentrations of reagent; under these conditions, a single essential tyrosine per molecule of transcortin seems implicated in cortisol binding. The absence of denaturation in modified transcortin was checked by circular dichroism spectra and polyacrylamide gel electrophoresis. Site specificity was demonstrated by full protection with cortisol against inactivation.

Binding Sites↗