Yves Boulanger

Purification et quelques proprietes de la phenylalanyl-tRNA synthetase de levure de boulangerie

Abstract Purification and some properties of phenylananyl-tRNA synthetase from bakers yeast Phenylalanyl-tRNA synthetase has been isolated from bakers yeast with a 600-fold purification. The different steps of the preparation are: (NH 4 ) 2 SO 4 precipitation of the 78 000 × g crude extract (between 50 and 65 % saturation), chromatography on DEAE-cellulose, CM-Sephadex C-50 and hydroxylapatite. The enzyme appears to be homogeneous on hydroxylapatite chromatography, sucrose gradient centrifugation and polyacrylamide gel electrophoresis. Molecular weight determinations by sucrose gradient centrifugation or equilibrium sedimentation studies give an average value of 220 000. Amino acid composition has been determined. No end group can be detected by the dansyl method. In the presence of either ATP and phenylalanine or tRNA Phe , the number of free thiol groups titrated with DTNB decreases. The enzyme is dissociated by 8 M urea or 1 % sodium dodecyl sulphate into two different equimolar components. The molecular weights of these 2 components were estimated to be 56 000 and 63 000, respectively, by polyacrylamide gel electrophoresis. The results suggest that the enzyme has a 4-subunits structure A 2 B 2 . The kinetics of the PP i -ATP exchange and aminoacylation reactions of tRNA Phe have been determined.

Complete purification and studies on the structural and kinetic properties of two forms of yeast valyl-tRNA synthetase

Two forms of bakers yease valyl-tRNA synthetase have been purified to apparent homogeneity by classical methods. It was demonstrated that one of the two forms of the enzyme originates from the other by proteolysis, the respective amounts of each form depending on the physiological state of the yeast. The species mainly isolated from exponential growing yeast cells is a monomer of 130,000 daltons molecular weight. In stationary phase cells or in commercial yeast the major species is a degraded monomer of 120,000 daltons molecular weight ; however when the purification is carried out in the presence of phenylmethyl-sulphonyl fluoride, or diisopropylfluorophosphate large amounts of the not - degreded monomer can be obtained. Of great practical usefulness is the fact that large amounts of the native enzyme can be obtained pure after only two chromatographic steps on DEAE-cellulose and hydroxylapatite. The kinetic constants for valine, ATP and tRNAVal were determined, as well as the optimum aminoacylation conditions. It was found that the specific activity of the nondegraded valyl-tRNA synthetase is higher than that of the proteolysed enzyme for the aminoacylation reaction. On the contrary, both forms have the same ATP-pyroposphate exchange activity. The amino acids composition of the native enzyme was established. The tryptic fingerprints of the two valyl-tRNA synthetases were studied. Essentially similar maps were obtained. The number of the spots in the fingerprints indicates that the enzymes contain a high proportion of repeated sequences.

Purification and some Properties of Bolesatine, a Protein Inhibiting In Vitro Protein Synthesis, from the Mushroom Boletus Satanas Lenz (Boletaceae)

The peptidic toxins of the Amanita species are well known (Wieland 1987). Proteinaceous toxins have also been reported to exist in mushrooms. Eilers and Nelson (1974) partially purified a toxin with an approximate Mr of 400000 from Chlorophyllum molybdites formerly named Lepiota morganii. More recently attention was drawn on the Boletaceae family, when cows died in Madagascar after ingestion of Boletus affinis Peck previously thought to be edible mushrooms. A toxic protein (Mr 22000) has been purified from these mushrooms in this laboratory (Razanamparany et al 1986). Its thermolability could explain the previously reported edibility. Furthermore this toxic protein, which was called bolaffinine, inhibits protein synthesis in hepatoma tissue culture cells and in rabbit reticulocyte lysates. The European boletus, Boletus satanas Lenz, although it is feared, has never been reported to be the cause of lethality in humans (Flammer 1980) or to contain any toxic protein (Czeczuga 1978; of Colceag et al 1984). Therefore it was of interest to know whether this boletus also contains a toxic protein comparable to the Madagascar Boletus affinis Peck.

γ-l-Glutamyl-l-aspartate induces specific deficits in long-term memory and inhibits [3H]glutamate binding on hippocampal membranes

gamma-L-Glutamyl-L-aspartic acid (gamma-LGLA) has been isolated from Datura stramonium; its structure has been determined and it was then synthesized. In male Swiss mice intraperitoneal administration of the natural peptide (125 nmol/kg) or of the synthetic peptide (25-2500 nmol/kg) 24 h after acquisition of a Y-maze avoidance task induced a dose-dependent deficit in retention performance 48 h later. gamma-LGLA had no effect on locomotor activity or emotional reactivity at the doses used. Separate or simultaneous administration of aspartate or glutamate (each at 250 nmol/kg) had no effect on learning retention, indicating that deficit induced by gamma-LGLA was specific to the peptide. gamma-LGLA impaired learning acquisition in a time-dependent manner when administered from 3 min to 24 h post-training, but had no effect when administered 3 days following acquisition. gamma-LGLA administered just after the training session did not affect retention performance during the first 3 h, but suppressed the retention improvement observed in control animals from 6 to 24 h after acquisition; this deficit was still evident 7 days after the treatment. gamma-LGLA partially inhibited L-[3H]glutamate binding on crude hippocampal or striatal membrane preparations; this inhibition was not observed on cerebellar membrane preparations. These results suggest a specific action of gamma-LGLA on excitatory amino acid systems which may be responsible for its effects on learning retention.

The glutaminyl-transfer RNA synthetase of Escherichia coli. Purification, structure and function relationship

Glutaminyl-tRNA synthetase from Escherichia coli has been purified to homogeneity with a yield of about 50%. It is a monomer of about 69 000 daltons. Arginyl and glutamyl-tRNA synthetases are also monomeric synthetases of molecular weight significantly lower than 100 000. In addition it is well known that these three synthetases require their cognate tRNA to catalyze the [32P]PPi-ATP exchange. Like arginyl-tRNA synthetase, but unlike glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase seems to contain some repeated sequences. Therefore no correlation can be established between the tRNA requirement of these synthetases for the catalysis of the isotope-exchange and the presence or the absence of sequence duplication. In the native enzyme four sulfhydryl groups react with dithiobisnitrobenzoic acid causing a loss of both the aminoacylation and the [32P]PPi-ATP exchange activities. The rate-limiting steps of the overall aminoacylation and its reverse reaction correspond, respectively, to the catalysis of the aminoacylation of tRNA Gln and of the the deacylation of glutaminyl-tRNA Gln. At acidic pH, glutaminyl-tRNA synthetase catalyzes the synthesis of the glutaminyl-tRNA Gln and its deacylation at significantly lower rates than the [32P]PPi-ATP exchange, indicating than glutaminyl-tRNA Gln cannot be an obligatory intermediate in this isotope exchange. These results suggest the existence of a two-step aminoacylation mechanism catalyzed by this enzyme.

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.

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.

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 Bakers 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.

Primary structure of aspartyl-tRNA synthetase from baker's yeast: tryptic and CNBr peptides

The cristallizable aspartyl-tRNA synthetase from Bakers yeast is a dimer made up of identical subunits (Mr 60,000). We report here the results of tryptic digestion and cyanogen bromide cleavage which enabled us to align two lon stretches of sequence of 106 and 111 amino acids, respectively.

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.