Affinity labelling of human transcortin.

The binding site of transcortin has been studied by using bromoacetyltestosterone and bromoacetylated derivatives of progesterone which were monohydroxylated at different positions of the steroid nucleus. Specificity of affinity labelling was demonstrated by the displad cortisol analog was added to a [3H]cortisol-transcortin complex solution. The binding site crevice was found to be very narrow in the vicinity of the A and B rings of steroid since 2alpha-hydroxyprogesterone, 6alpha- or 6beta-bromoacetoxyprogesterone and dexamethasone could not displace bound cortisol. A specific affinity labelling was obtained with 11alpha-bromoacetoxyprogesterone, 16alpha-bromoacetoxyprogesterone and 17beta-bromoacetyltestosterone. The results of the affinity labelling by these hormone analogs suggested that one methionine and one histidine residues were located within the active site:methionine might interact with the 11beta-hydroxyl group and histidine with the 20 keto group of cortisol.

The covalent structure of dog myoglobin.

The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.

Comparison of the amino acid sequence of pig heart myoglobin with other ungulate myoglobins.

The primary structure of pig heart myoglobin has been established by study of the tryptic peptides of whole globin and by analysis of the fragments obtained by CNBr cleavage. Thermolysin and chymotrypsin digestion were used to determine the sequence of the M fragment (56-131). Automatic Edman degradation of whole globin and of the M fragment completed the sequence of pig myoglobin. Comparison with other ungulates shows that pig myoglobin is far from other artiodactyls previously studied (ox and sheep) and close to the eutherian ancestral chain.

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.

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.

A redox function for the Calpha2 domain of IgA immunoglobulin.

IgA1 populations were reduced over a range of dithiothreitol concentrations and the products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping. The study showed that IgA was more resistant to reduction than other immunoglobulin classes (IgG, IgM). SDS-polyacrylamide gel electrophoresis showed that intersubunit bond and H-L bond were the most labile disulfide in polymeric and monomeric IgA, respectively. Peptide mapping revealed that the itrachain disulfide of the C alpha 2 domain was more or equally sensitive to the reduction than H-L and intersubunit bonds. Electrochemical experiments demonstrated that this bond had redox properties and the possibility involving disulfide exchange is discussed.

Hydrodynamic characterization and DNA-binding properties of the liganded and unliganded forms of the retinoic-acid receptor alpha from human HL-60 cells.

The hydrodynamic parameters of the retinoic-acid receptor from human myeloblastic leukemia HL-60 cells were accurately investigated. The ligand-bound retinoic-acid receptor (RAR) has a Stokes radius of 3.5 nm when analyzed by size-exclusion chromatography. A 53-kDa protein was detected by Western blot analysis using a polyclonal antibody directed against the F domain of hRAR alpha, in the fractions containing the 3.5-nm complex. Fractionation of a crude nuclear extract from HL-60 cells, untreated with retinoic acid, yielded antibody-revealed material with Stokes radii ranging from 3.5 nm to 6 nm. From the hydrodynamic data, a molecular mass of 52 kDa was calculated for the liganded receptor, whereas no precise value could be deduced for the unliganded receptor form, since it dissociates rapidly into the 3.5-nm form. Gel-retardation experiments showed that the 3.5-nm form of hRAR alpha bound specifically to DNA, whereas binding of the unliganded receptor form was sharply reduced. These findings suggest that the unliganded inactive receptor form dissociates upon ligand binding and acquires a ligand-dependent DNA-binding activity.

Localization of J-chain and interchain disulfide bonds in a human F(c)5mu-like fragment.

The inter H-H cysteinyl peptides and the localization of the J-chain were studied in a human F(c)5mu-like fragment. The latter was found to be built up by non-covalent association of molecular forms of 140 000, 95 000 and 70 000 dalton subunits. The trimeric, dimeric and monomeric forms were obtained from gradual reduction by dithiothreitol of the major component of 140 000 daltons, thus confirming the tetrameric nature of this subunit. The latter was found to result from the association of both components of the 70 000 dalton subunit, with the participation of the inter H-H subunit bridge. Structural analysis of the labelled peptides obtained by partial reduction and alkylation showed the presence of the intersubunit disulfide bridge and of the inter heavy-heavy chain bridge of the C-terminal region, and the absence of the heavy-heavy chain bridge of the hinge region. The sequence of these peptides is identical to the sequences of the corresponding peptides of normal mmu-chains. The J-chain, which was covalently linked to this F(c)5mu-like fragment, was found to be predominantly associated within the 95 000 dalton subunit. The results showed that the J-chain was linked in the protein as a "clasp" within a single subunit and not between two subunits.

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.

A protein kinase C-dependent activity modulates retinoic acid-induced transcription.

The retinoic acid receptors (RARs) and retinoid X receptors, which are members of the nuclear receptor family, mediate the effects of vitamin A derivatives on cellular growth and differentiation. The protein kinase C isozyme family also controls these processes in response to extracellular stimuli. We have investigated the relationship between these two signal transducing pathways using gene transfer techniques. We show that selective inhibition of protein kinase C (PKC) and its depletion by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate lead to the loss of ligand-dependent transcription of an RA-inducible promoter. The effect of the depletion in cellular PKC could be counteracted by overexpression of PKC alpha and is directly correlated to the loss of the DNA-binding activity of complexes containing the human RAR alpha (hRAR alpha). Indirect immunofluorescence studies demonstrated an altered subcellular localization of hRAR alpha. However, direct in vitro phosphorylation of hRAR alpha by PKC diminished its ability to form heterodimeric or homodimeric complexes on a retinoic acid response element, suggesting that the DNA-binding capacity of hRAR alpha in intact cells is indirectly controlled by a PKC-dependent mechanism. Thus our observations establish a functional link between the PKC and retinoid pathways, which are generally considered to have antagonistic activities on differentiation processes.

Biochemical and immunological evidence that an acidic domain of hsp 90 is involved in the stabilization of untransformed glucocorticoid receptor complexes.

Polyclonal antibodies (AS 232-266) have been raised against the 232-266 amino acid sequence of the mouse hsp 84. This sequence possesses 54% acidic residues. AS 232-266 react with both the denatured and the free native murine hsp 84, but not with the bound hsp 84 present in the untransformed glucocorticoid receptor complexes (GR). Both AS 232-266 and peptide 232-266 were shown to decrease [3H]dexamethasone binding by GR. Moreover synthetic peptide 232-266, when added to 7 nm untransformed GR, convert them into 5 nm hsp 84-free GR. Taken together these data suggest that the acidic 232-266 sequence of hsp 84 is involved in the stabilization of the hsp 84-GR interaction, which is known to result in 7 nm complex formation and in GR ligand binding activity improvement. Both peptide 232-266 and AS 232-266 destabilize this interaction.

Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity.

The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.

Developmental aspect of phenylalanine hydroxylase in the rat -- hormonal influences.

Interpretations of the development of phenylalanine hydroxylase (PAH) in rat liver have been controversial, and the mechanism of ontogenic changes have not yet been elucidated. Fetal PAH activity at a gestational age of 21 days appeared to reach 32% that of adult male level at birth. The in vivo effectiveness of fetal PAH activity was correlated with enhancement of blood tyrosine, while amino-transferase activity appeared only after birth. No sex difference was noted in weaning rats, whereas, in adult females, PAH activity was only 42% that of males. Investigating hormonal influences on liver PAH activity we noted no change of enzyme activity following hydrocortisone, ACTH and estradiol treatment. However, 4 days of testosterone treatment in weaning female rats increased PAH activity (X1.7). Therefore, testosterone could explain increased PAH activity in adult males.

[Specificity of thermolysin action on dog myoglobin]. / Spécificité d'action de la thermolysine sur la myoglobine de chien

The proteolytic specificity of thermolysin has been studied by quantitative analysis of an enzymic digest of dog myoglobin. Results confirm main specificities of thermolysin towards Phenylalanine, Isoleucine, Leucine or Tyrosine bonds; the influence of neighbourhood was also determined and the conclusions are in a good agreement with the known structure of the active site of thermolysin.

Human serum hemopexin: direct evidence for change of its isoelectric point upon heme binding. A new serum protein fractionation.

Human serum was submitted to a one step displacement-ligand exchange chromatography. Displacement removed serum albumin and part of gamma-globulins. Ligand exchange furnished an enriched heme-hemopexin fraction. An original, non denaturing human heme-hemopexin preparation is proposed.

[Characterization and physico-chemical properties of human transcortin]. / Caractérisation et propriétés physico-chimiques de la transcortine humaine

The molecular weight of human transcortin, calculated from the sedimentation coefficient, was found to be 49,500, thus slightly lower than previously reported values. After purification, human transcortin trended to polymerize rapidly, with participation of both non covalent bonds and one disulfide bridge per dimer. The physicochemical parameters, the amino-acid and carbohydrate composition were determined; its stability was studied under different conditions. Preliminary structural studies showed that the N-terminal sequence of the polypeptide chain was: Met-Asp-Pro-Asn-Ala-Ala-Tyr-Val and that the C-terminal amino acid was leucine.

A new experimental model of hyperphenylalaninemia in rat. Effect of p-chlorophenylalanine and cotrimoxazole.

A new experimental model of hyperphenylalaninemia was proposed. Combination of p.chlorophenylalanine, strongly inhibitor of phenylalanine hydroxylase, and cotrimoxazole, presumably inhibitor of dihydropteridine reductase, produced a good inhibition of phenylalanine hydroxylation in vivo. Thus phenylalaninemia reached values similar to those found in PKU patients, without administration of excess phenylalanine. Tyrosine concentrations remained near the control values and a phenylketonuria occurred.

[Sequential Edman degredation]. / Dégradation récurrente d'Edman.

Since Edman's first publication in 1950, the stepwise degradation of proteins and peptides is universally performed by protein chemists. We extensively reviewed the different manual degradations. We take two examples of manual degradation: a semi-micromethod and a micromethod in order to illustrate the evolution of manual degradation. The "dansyl-Edman" procedure proposed by Hartley in 1963 completes the manual N-terminal determination of peptides. We describe the different procedures of identification of PTH-amino acids: paper chromatography, thin layer chromatography, gas chromatography and liquid chromatography under high pressure and various modified Edman degradation procedures. Possibilities and limits of the liquid phase Sequenator of Edman reported in 1967 and the solid phase Sequencer of Laursen reported in 1971 are also considered in detail.

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.

Phenylalanine analogues as inhibitors of phenylalanine-hydroxylase from rat liver. New conclusions concerning kinetic behaviors of the enzyme.

The conversion of phenylalanine to tyrosine is catalysed by phenylalanine-hydroxylase. The substrate phenylalanine shows two effects: (1) allosteric transition at low phenylalanine concentrations, (2) excess substration inhibition. The molecular structure of phenylalanine-hydroxylase has not yet been elucidated. However, a tetrameric structure has been proposed. The Kinetic analysis with respect to substrate analogues suggest the existence of three types of sites on each protomer: (1) a catalytic site, (2) a non-competitive inhibitory site, (3) a positive cooperative site. Use of the enzyme's natural cofactor, tetrahydrobiopterin, has been emphasized to ensure good interpretation of the kinetic results of the phenylalanine-hydroxylase effectors.