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J Baudier

Publications and source records attributed to J Baudier.

At least 55 records · Page 3Linked to original sources
PubMed1986

Bimane- and acrylodan-labeled S100 proteins. Role of cysteines-85 alpha and -84 beta in the conformation and calcium binding properties of S100 alpha alpha and S100b (beta beta) proteins.

Bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein dimers were labeled with the sulfydryl-specific fluorescent probes monobromo(trimethylammonio)bimane (bimane) and 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) at cysteines-85 alpha and -84 beta. The conformation and fluorescence properties of the S100 proteins derived were studied by means of anion-exchange chromatography on a Mono Q column using a fast protein chromatography system and fluorescence intensity, maximum emission wavelength, and polarization measurements. Spectroscopic studies on the intrinsic absorption and fluorescence properties of S100 alpha alpha and S100b proteins chemically modified on cysteines-85 alpha and -84 beta with iodoacetamide completed this study. Several arguments suggest that the alkylated S100 proteins undergo conformational changes that are mainly characterized by the destabilization of the quaternary protein structure, which provokes a slow dimer-monomer equilibrium at high protein concentrations and induces total subunit dissociation at low ones. Calcium binding studies on bimane-S100 alpha alpha and -S100b proteins showed that alkylated proteins had a much higher calcium binding affinity than native protein and that the antagonistic effect of KCl on calcium binding was much less pronounced. These results confirmed our previous observations that the affinities of calcium binding sites II alpha and II beta in S100 proteins are highly dependent on protein conformation [Baudier, J., & Gerard, D. (1986) J. Biol. Chem. 261, 8204-8212].

2-Naphthylamine↗
PubMed1986

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein.

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.

Animals↗
PubMed1986

Ions binding to S100 proteins. II. Conformational studies and calcium-induced conformational changes in S100 alpha alpha protein: the effect of acidic pH and calcium incubation on subunit exchange in S100a (alpha beta) protein.

A rapid separation method for bovine brain S100 alpha alpha, S100a, and S100b protein using fast protein liquid chromatography on a Mono Q column and its application in preparation of a large amount of S100 alpha alpha protein are described. The conformation of S100 alpha alpha in the metal-free forms as well as in the presence of calcium were studied by UV absorption, circular dichroism, intrinsic fluorescence, sulfhydryl reactivity, and interaction with a hydrophobic fluorescent probe. The alpha-subunit appears to have nearly identical conformation in S100 alpha alpha and S100a protein dimers. We also confirmed that only the alpha-subunit exposes hydrophobic domains to solvent in the presence of calcium and that cysteine residues exposed upon Ca2+ binding to S100 proteins correspond to Cys 85 alpha and Cys 84 beta. Incubation of S100a with calcium and KCl proved that calcium binding to the putative calcium-binding sites (site I alpha, I beta) triggers a time- and temperature-dependent conformational change in the protein structure which decreases the antagonistic effect of KCl on calcium binding to sites II alpha and II beta and provokes subunit exchanges between protein dimers and the emergence of S100 alpha alpha and S100b (beta beta) proteins. Dynamic fluorescence measurements showed that incubating calcium at high S100a protein concentrations (greater than 10(-5) M) induces an apparent slow dimer-monomer equilibrium which might result in total subunit dissociation at lower protein concentrations. The effect of acidic pH on subunit dissociation in S100a protein (Morero, R. D., and Weber, G. (1982) Biochim. Biophys. Acta 703, 231-240) arises from conformational changes in the protein structure that are similar to those induced by Ca2+ incubation.

Animals↗
PubMed1986

Evidence that S100 proteins regulate microtubule assembly and stability in rat brain extracts.

Microtubule re-assembly in rat brain extracts was inhibited by antibodies to S100 proteins. Anti-S100 antibodies caused an increase in the cold-stability of microtubules and this effect was abolished by the presence of short lengths of microtubules formed under control conditions. Anti-S100 antibodies had no effect on the stimulation of assembly or the increase in microtubule stability caused by low zinc concentrations. Addition of exogenous S100a and S100b to brain extracts had different effects on assembly; S100a caused an inhibition of assembly while S100b stimulated the early phase of assembly. The data suggest that endogenous S100b is involved in the regulation of microtubule assembly in brain extracts.

Animals↗
PubMed1986

Immunocytochemical localisation of caligulin-like immunoreactivity in rat tissues.

The cellular localisation of caligulin-like immunoreactivity (caligulin-LI) in rat central and peripheral tissues was studied using antibodies against bovine brain caligulin raised in rabbits. Both immunofluorescence and immunoperoxidase techniques were used to demonstrate caligulin-LI in paraformaldehyde fixed tissues. Certain neurons in the cerebral cortex, basal ganglia and brain stem contained caligulin-LI. In the cerebellum a majority of Purkinje cells were labelled with immunoreactive material localised to dendrites, perikarya and axons. In the gastrointestinal tract some neurons in Auerbach's and Meissner's plexa contained caligulin-LI. Ligation of the sciatic nerve caused accumulation of immunoreactive material both proximal and distal to the crush. A non-neuronal localisation of caligulin-LI could also be demonstrated, e.g., in parts of the renal tubular system, in the islets of Langerhans and in certain enteroendocrine cells. The specific localisation of caligulin-LI in some but not all neurons of the rat brain and gastrointestinal tract suggests a specific function of caligulin in central and peripheral nervous mechanisms.

Animals↗
PubMed1985

Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain.

A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.

Animals↗
PubMed1985

Rat brain S100b protein: purification, characterization, and ion binding properties. A comparison with bovine S100b protein.

We purified to homogeneity rat brain S100b protein, which constitutes about 90% of the soluble S100 protein fraction. Purified rat S100b protein comigrates with bovine S100b protein in nondenaturant system electrophoresis but differs in its amino acid composition and in its electrophoretic mobility in urea-sodium dodecyl sulfate-polyacrylamide gel with bovine S100b protein. The properties of the Ca2+ and Zn2+ binding sites on rat S100b protein were investigated by flow dialysis and by fluorometric titration, and the conformation of rat S100b in its metal-free form as well as in the presence of Ca2+ or Zn2+ was studied. The results were compared with those obtained for the bovine S100b protein. In the absence of KCl, rat brain S100b protein is characterized by two high-affinity Ca2+ binding sites with a KD of 2 X 10(-5) M and four lower affinity sites with KD about 10(-4) M. The calcium binding properties of rat S100b protein differ from bovine S100b only by the number of low-affinity calcium binding sites whereas similar Ca2+-induced conformational changes were observed for both proteins. In the presence of 120 mM KCl rat brain S100b protein bound two Zn2+-ions/mol of protein with a KD of 10(-7) M and four other with lower affinity (KD approximately equal to 10(-6) M). The occupancy of the two high-affinity Zn2+ binding sites was responsible for most of the Zn2+-induced conformational changes in the rat S100b protein. No increase in the tyrosine fluorescence quantum yield after Zn2+ binding to rat S100b was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein.

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.

Animals↗
PubMed1984

Purification, characterization and ion binding properties of human brain S100b protein.

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.

Binding Sites↗
PubMed1984

A subnanosecond-pulse fluorometric study of the CA2+ and MG2+ induced conformational changes on S-100a protein.

The fluorescence parameters of the single tryptophan residue (Trp 90 alpha) in S-100a (alpha beta) protein have been studied by steady state fluorimetry and by subnanosecond fluorescence decays excited by pulsed-picosecond laser system. At pHs 7.1 and 8.5, double exponential decays were consistently observed. At both pHs, Ca2+ and to a less extent Mg2+ ions proved to modify the percentage contribution of the two decaying species. The interest of the finding is discussed.

Animals↗
PubMed1984

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands.

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.

Animals↗
PubMed1984

S-100 protein in melanocytic tumors. An immunohistochemical investigation of benign and malignant melanocytic tumors and metastases of malignant melanoma and a characterization of the antigen in comparison to human brain.

An immunohistochemical investigation regarding the presence of S-100 protein in benign and malignant, primary and metastatic melanocytic tumors is reported. The studied series consisted of 15 benign nevi, 3 blue nevi, 4 juvenile melanomas, 1 balloon cell nevus, 30 primary malignant melanomas of skin, mucous membranes and conjunctiva and 30 metastatic malignant melanomas. The immunohistochemical analysis showed positive staining for S-100 protein within the majority of the tumor cells in all benign tumors examined, except the balloon cell nevus, as well as in all the primary and metastatic malignant melanomas, including low-differentiated epitheloid or spindle-cell types without demonstrable melanin pigment. The results indicate that S-100 protein is a valuable marker for melanocytic tumors, especially in the fairly frequent dilemma of malignant melanoma presenting as a solitary non-pigmented metastasis of uncharacteristic light-microscopic appearance, and without known primary tumor. A characterization and quantification of the S-100 protein immunoreactivity in 5 metastatic malignant melanomas is presented. Using immuno-electrophoresis, the presence of 3 antigenic S-100 determinants was demonstrated within homogenates from the malignant melanomas, including the previously characterized S-100 A (alpha/beta) and S-100 B (beta/beta), and suggesting the presence of a hitherto undescribed variant of S-100 protein, possibly consisting of 2 alpha-subunits. Using rocket immuno-electrophoresis, the amount of S-100 protein was estimated.

Antigens, Neoplasm↗
PubMed1983

The effect of S-100a and S-100b proteins and Zn2+ on the assembly of brain microtubule proteins in vitro.

The homologous proteins S-100a and S-100b affect the microtubule system in a distinctly different way in the presence of low molar ratios of Zn2+. Assembly of brain microtubule proteins can be almost completely inhibited and rapid disassembly can be induced by low molar amounts of S-100b in the presence of low molar ratios [2-4] of Zn2+. Higher molar ratios per S-100b (greater than 4) potentiate the general Zn2+ effect, promoting the formation of sheets of microtubules. However, the effect of S-100a is quite different, no inhibition of assembly can be observed and the presence of S-100a seems to protect the microtubule proteins against the effect of Zn2+ by chelating the Zn2+ and decreasing the free metal-ion concentration. S-100a or S-100b cannot bind to the microtubule polymer-form, either in the absence or in the presence of Zn2+.

Animals↗
PubMed1983

Zinc ion binding to human brain calcium binding proteins, calmodulin and S100b protein.

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn2+ binding sites, with KD ranging from 8.10(-5)M to 3.10(-4)M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. KD = 10(-7) - 10(-6)M. We suggest that S100b protein should no longer be considered only as a "calcium binding protein" but also as a "zinc binding protein", and that Zn2+ ions are involved in the functions of the S100 proteins.

Brain↗
PubMed1983

The S100-b protein: tyrosine residues do not exhibit an abnormal fluorescence spectrum.

The beta subunit of the bovine brain S100-b protein (beta beta) lacks tryptophyl residue but contains one tyrosine. Our experiments show that this protein is characterized by a typical tyrosine fluorescence spectrum, with a maximum at 303 nm. Identical fluorescence properties were found for the rat brain S100-b protein. Comparison with the fluorescence spectrum of the bovine brain S100-a' protein (alpha'beta), which contains a tryptophan residue in the alpha' subunit, enables us to demonstrate that the recent report describing an abnormal fluorescence spectrum for the bovine brain S100-b protein may result from a contamination of the S100-b by the S100-a' protein.

Animals↗
PubMed1983

Bovine brain S100 proteins: separation and characterization of a new S100 protein species.

Three S100 protein species (S100a, S100b, S100a') have been purified from bovine brain using a modification of standard preparative methods. A higher yield for each protein was obtained at the last separation step. Characterization by urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis, UV absorption spectra, and fluorescence parameters provided evidence of a new tryptophan-containing S100 protein called S100a', which exhibits, as S100a and S100b, the properties of a Ca2+ binding protein.

Animals↗