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Biomedical subjects

Y Boulanger

Publications and source records attributed to Y Boulanger.

At least 73 records · Page 4Linked to original sources
PubMed1986

Use of dialysis fibers for the study of aerobic cells.

The suitability of a perifusion system for the study of renal tubules trapped in different types of dialysis fibers has been investigated. First, the equilibration times across the fiber membranes were estimated by following the penetration of the dysprosium tripolyphosphate shift reagent inside the fibers using 23Na nuclear magnetic resonance. These times ranged from less than 1 min for microfibers to 30 min for some large diameter fibers. When cortical tubules were introduced into cuprophan dialysis microfibers (200 microns internal diameter) and perifused by rapid recirculation of cell culture medium, metabolic viability, as determined by a linear glucose and ammonia production from glutamine and lactate, was found to exceed 10 (dog) or 3 h (rat), an unprecedented time period for such structures. As evidenced by the sensitivity to furosemide, the lumen of thick ascending limb tubules was kept open by including 50 mM mannitol in the perifusate. Fibers with large internal diameters (1100 microns) were found to be less suitable owing to the slow equilibration rates with the intrafiber compartment. All types of fibers showed minimal line-broadening effects on the 31P nuclear magnetic resonance signals.

Animals↗
PubMed1985

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus.

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1985

An improved perifusion system for NMR study of living cells.

Isolated cells can be kept in steady-state conditions for very long periods of time when inserted into dialysis microfibers and perifused with a nutrient-containing solution. Using this system, stable 31P NMR spectra have been obtained on dog kidney tubules for a period of 13 h.

Animals↗
PubMed1985

A home-made solid-phase sequencer operating at the nanomole level.

The building and functioning of a fully automated solid-phase sequencer is described. The peptide is coupled via its alpha-carboxyl end to activated glass beads and successively reacted with Chang's and Edman's reagents. All operations are electronically controlled by the automated programmer. All components necessary to build the machine are commercially available. This sequencer has been used at a nanomole level in the final phase of a protein sequence determination. The overall cost as well as the sensitivity and efficiency of the final product compare favourably to those of commercial machines.

Alkylation↗
PubMed1985

Measurement of a wide range of intracellular sodium concentrations in erythrocytes by 23Na nuclear magnetic resonance.

The accuracy of the 23Na nuclear magnetic resonance (NMR) method for measuring the sodium concentration in erythrocytes was tested by comparing the NMR results to those obtained by emission-flame photometry. Comparisons were made on aqueous solutions, hemolysates, gels, ghosts, and intact erythrocytes. The intra- and extracellular 23Na NMR signals were distinguished by addition of the dysprosium tripolyphosphate [Dy(PPP)7-2] shift reagent to the extracellular fluid. The intra- and extracellular volumes of ghosts and cells were determined by the isotope dilution method. Our results indicate that greater than 20% of the intracellular signal remains undetected by NMR in ghosts and cells. When the cells are hemolyzed, the amount of NMR-detectable sodium varies depending on the importance of gel formation. In hemolysates prepared by water addition, the NMR and flame photometry results are identical. The loss of signal in ghosts, cells, and undiluted hemolysates is attributed to partial binding of the Na+ ion to intracellular components, this binding being operative only when these components exist in a gel state. In a second part, 31P NMR was used to monitor the penetration of the shift reagent into the cells during incubation. Our data demonstrate that free Dy3+ can slowly accumulate inside the red cell.

Animals↗
PubMed1985

The complete amino acid sequence of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

The crystallizable cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63 000). Its primary structure was established using peptide sequences from four different digests of the native and citraconylated enzyme with trypsin, cyanogen bromide and staphylococcal protease. The oligonucleotide sequence of the structural gene was used as a template for the final alignment of the various peptides in the correct order.

Amino Acid Sequence↗
PubMed1985

Amino acids in normal and diabetic ducks.

The transient diabetes, observed in ducks after subtotal pancreatectomy, induces hyperglycaemia and hyperamino-acidaemia. Threonine and alanine are quantitatively the most important amino acids in both normal and diabetic animals, suggesting a particular role of threonine in amino acid metabolic pathways in the duck. The hyperaminogenic role of the decreased insulin levels, and the neoglucogenic effect of a low, but not negligible, glucagon secretion, during diabetes, are discussed.

Amino Acids↗
PubMed1984

NMR analysis of the structure and metal sequestering properties of metallothioneins.

113Cd-NMR studies have been used to elucidate the structure of the metal-binding sites in mammalian and invertebrate ( Scylla serrata) metallothioneins (MTs). Chemical shift data have shown that all Cd ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. Homonuclear decoupling experiments have shown that the 7 g-atoms of metal bound per mole of mammalian protein are located in a three- and a four-metal cluster while the 6 g-atoms of metal in the invertebrate MT are located in two three-metal clusters. The different metal binding affinities of the two mammalian clusters have been determined by 113Cd-NMR. The three-metal cluster prefers Cu greater than Zn greater than Cd whereas exactly the reverse order applies in the four-metal cluster. Proteolytic cleavage of the protein produced a 32-residue fragment which contained the four-metal cluster and demonstrated the presence of two separate domains in the protein. 500 MHz 1H-NMR has been employed to elucidate the arrangement of these metal clusters in the tertiary structure of the protein. The 1H resonances were assigned from their scalar and dipolar connectivities obtained from extensive one and two-dimensional NMR experiments. A specific application of 2D correlation spectroscopy ( COSY ) to the assignment of the 1H resonances in crab MT-1 is discussed. A molecular model, representing the three-dimensional solution structure of this protein, has been constructed based on an analysis of all these data. Detailed structural features of this model are discussed, with particular emphasis on their relationship to the function and evolution of the protein.

Animals↗
PubMed1983

Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase.

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated form an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the alpha- and beta-subunits composing the native alpha 2 beta 2 enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.

Amino Acid Sequence↗
PubMed1983

Glycosylation of yeast aspartyl-tRNA synthetase. Affinity labelling by glucose and glucose 6-phosphate.

Several lines of evidence establish that the crystallizable aspartyl-tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl-tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [14C]glucose or [14C]glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein. Indeed immunoprecipitation of [14C]glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl-tRNA synthetase.

Affinity Labels↗
PubMed1983

Reversed-phase liquid chromatography of peptides for direct micro-sequencing.

Tryptic and cyanogen bromide peptides derived from yeast aspartyl-tRNA synthetase and from Escherichia coli ribosomal proteins were separated by reversed-phase liquid chromatography, employing volatile buffers of low ionic strength. The conditions used allow the performance of micro-sequencing without desalting or extensive lyophilization, and can therefore be applied to peptide mixtures containing hydrophobic fragments which tend to precipitate. To prevent losses of peptides, direct ultra-violet detection of the peptides was preferred, to detection by post-column derivatization with an additional stream splitting device. Preparative separations were performed with 5-10 nmol of peptide mixture; analytical runs were made with 5-10 micrograms of protein hydrolysate.

Amino Acid Sequence↗
PubMed1983

Model for mammalian metallothionein structure.

The results of physicochemical studies of mammalian metallothioneins are summarized and used to propose a model of the protein. The primary structures of all mammalian metallothioneins are very homologous; there are 38 invariant residues and 20 of them are cysteines. The results of UV and CD optical studies indicated that all 20 cysteines are involved in the ligation of 7 mol of metal per mol of metallothionein and that the protein does not contain any alpha-helix structure. A theoretical analysis by the Chou-Fasman method has predicted 11 beta-bends, each one involving at least one cysteine residue. The most significant structural data, provided by 113Cd NMR, demonstrated that the 7 mol of bound Cd2+ are arranged in two separate metal clusters, one containing four metal ions and the other containing three, with all Cd2+ tetrahedrally coordinated to cysteine thiolate ligands. The 11 cysteine residues of the carboxyl-terminal portion of the metallothionein chain (residues 30-61) are ligated to the 4-metal cluster as shown by 113Cd NMR of this enzymatically cleaved fragment. The remaining cysteine residues from the amino-terminal polypeptide portion (residues 1-29) form the 3-metal cluster. Such a division of the chain is consistent with the presence of an intron in the mouse metallothionein-1 gene corresponding to residue 32 in the polypeptide chain. A two-domain molecular model has been constructed based on an analysis of all the available data and is described in detail. The accuracy of this model was tested by 1H NMR at 500 MHz and the data are in agreement with our proposed structure.

Amino Acid Sequence↗
PubMed1983

Primary structure of the Saccharomyces cerevisiae gene for methionyl-tRNA synthetase.

The sequence of a 5-kilobase DNA insert containing the structural gene for yeast cytoplasmic methionyl-tRNA synthetase has been determined and a unique open reading frame of 2,253 nucleotides encoding a polypeptide chain of 751 amino acids (Mr, 85,500) has been characterized. The data obtained on the purified enzyme (subunit size, amino acid composition, and COOH-terminal sequence) are consistent with the gene structure. The protein sequence deduced from the nucleotide sequence reveals no obvious internal repeats. This protein sequence shows a high degree of homology with that of Escherichia coli methionyl-tRNA synthetase within a region that forms the putative methionyl adenylate binding site. This strongly suggests that both proteins derive from a common ancestor.

Amino Acid Sequence↗
PubMed1982

113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure.

A 32-residue polypeptide fragment, designated alpha I, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid composition of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of alpha I-fragment. Four 113Cd resonances were observed, three of which had identical chemical shifts to those assigned to the four-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chemical shift difference between the remaining resonance assigned to the four-metal cluster in the intact protein can be explained to result from the removal of the NH2-terminal polypeptide fragment containing the three-metal cluster. These results provide unambiguous evidence for the two-domain structure of metallothionein, containing a separate three- and a four-metal cluster.

Animals↗
PubMed1982

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases from Euglena gracilis. Comparative study of their structural properties.

Chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetases purified from Euglena gracilis show a monomeric structure. The molecular weights of the two valyl-tRNA synthetases are identical (126,000) while those of the leucyl-tRNA synthetases are different (100 000 for the chloroplastic and 116 000 for the cytoplasmic enzyme). The tryptic maps and the amino acid compositions reveal differences between the chloroplastic valyl- and leucyl-tRNA synthetases and their cytoplasmic homologues. These results suggest that a chloroplastic aminoacyl-tRNA synthetase and its cytoplasmic counterpart are coded for by distinct genes.

Amino Acids↗
PubMed1982

Affinity labelling of yeast phenylalanyl-tRNA synthetase with a 3'-oxidised tRNAPhe. Isolation and sequence of the labelled peptide.

Yeast phenylalanyl-tRNA synthetase was specifically labelled with a 3'-oxidised tRNAPhe. Stoichiometric inactivation was achieved with the incorporation of 2 mol oxidised tRNA Phe/mol enzyme which corresponds exactly to the stoichiometry of tRNA binding. The labelled peptide has been isolated using a quick chromatographic procedure which can be applied to any covalent complex formed between a tRNA and an aminoacyl tRNA synthetase. The isolated peptide (18 amino acids) was found to encompass the unique cysteine sequence of the smaller beta subunit of the enzyme.

Affinity Labels↗