Julie Labouesse

Purification of tRNATrp, tRNAVal, and partial purification of tRNAIle and tRNAMetf from beef liver

tRNATrp from beef lever has been purified by classical chromatographical methods. Total tRNA, prepared on a large scale (total aminoacid acceptance 1280 pmol/A260 unit) was submitted to chromatography on benzoylated-DEAE cellulose, then DEAE Sephadex. The major species accepting tryptophan issued from the second chromatography was aminoacylated with [14C] tryptophan and chromatographed on benzoylated-DEAE cellulose. The tRNA carrying the radioactive label was eluted in the ethanolic region. After stripping, the resulting tRNATrp has an acceptance of 1800 pmol/A260 unit. No isoacceptors could be demonstrated by chromatography of the pure species on RCP 5 in 6 M urea. The yield in pure tRNATrp was currently in the range of 25 to 30 percent of the total tryptophan acceptance of the starting curde tRNA.

Primary structure of bovine liver tRNATrp.

Purified tRNATrp from bovine liver, accepting 1700 pmol tryptophan per A260nm unit, was completely digested with pancreatic ribonuclease and T1 ribonuclease. The sequences of the resulting oligonucleotides were determined and the primary structure of the tRNA was deduced. These analyses showed numerous incomplete post-transcriptional modifications, and several positions heterogenously occupied by two different nucleotides, which lead us to think that in bovine liver there exist a mixture of several tRNATrp.

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

A quenched-flow study of a receptor-triggered second messenger response: Cyclic AMP burst elicited by isoproterenol in C6 glioma cell membranes

Fast kinetic studies of cAMP accumulation in C6 cell membranes show a burst of cAMP after β‐adrenergic receptor stimulation by isoproterenol. This burst is no longer observed when the ATP present in membrane preparations is hydrolyzed, but can be restored by their preincubation in the presence of ATP‐Mg. The size of the burst is much larger than the number of β‐adrenergic receptors and is of the same order of magnitude as the value reported for G proteins. Further characterization of the burst will allow studies of the functional interaction of receptor‐adenylate cyclase components in C6 membranes.

Influence d'hormones ovariennes et thyroïdiennes sue deux décarboxylase d'acides aminés dans le foir du rat. Mise en évidence de l'existence de deux régulations indépendantes

Abstract Injections of physiological dosesof oestradiol strongly diminish L -cysteinsulphinic acid decarboxylation and slightly increase L -3,4-dihydroxyphenylalanine decarboxylation in the liver of thyroidectomized rats. The injection of physiological doses of thyroxine reduces both decarboxylations in the female rat following ovarietomy and thyroidectomy. Thus for two enzymes studied there are two independent types of hormonal regulation.

Comparaison de la régulation, in vivo, par les hormones ovariennes du taux de deux décarboxylasesd'acides aminés dans le foie du rat

Abstract A comparative study was made of the influence in vivo of ovarian hormones on two amino acid decarboxylases in rat liver. Oestradiol, while markedly reducing the rate of L -cysteinsulphinic acid decarboxylation, generally does not appreciably affect the level of dopadecarboxylase. However, in certain groups of animals oestradiol slightly augments the activity of this enzyme. No action of oestradiol on cysteinsulphinic acid decarboxylase could be demonstrated in vitro.

Primer tRNATrp enhances the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate

Abstract In the presence of tRNA Trp the inhibition of avian myeloblastosis virus reverse transcriptase by pyridoxal-5′-phosphate is greatly enhanced. This effect of tRNA is specific. While tRNA beef Trp (the primer of the in vitro DNA synthesis with 35 S viral RNA as template) gives the maximal effect, tRNA beef Val will not affect the enzyme activity. In the presence of tRNA beef Trp two additional lysines are titrated with pyridoxal-5′-phosphate as measured by reduction of the enzyme-pyridoxal-5′-phosphate complex with tritiated NaBH 4 . The effect of tRNA is dependent on the presence of the β subunit of avian myeloblastosis virus reverse transcriptase, since the enzymatic activity of the α subunit, although inhibited by pyridoxal-5′-phosphate, is not affected by tRNA.

Influence comparée du chlorure de sodium et du tween 80 sur l'activité enzymatique de la carboxypeptidase a du pancréas☆

Resume Letude cinetique de lhydrolyse par la carboxypeptidase de quelques uns de ses substrats, en presence soit de Tween 80, soit de chlorure de sodium, a montre quentre 25° et 37° la constante de Michaelis est tois plus faible en presence de Tween quen presence de sel, et ceci quel que soit le substrat. Par contre, a une meme temperature, les constantes de vitesse de decomposition du complexe enzyme-substrat, ainsi que les constantes thermodynamiques de lactivation de de complexe sont tres voisines quelles que soient les conditions. Letude de linfluence de substances tensioactives autres que le Tween 80, a montre que les groupes de substances tensioactives non ioniques et cationiques sont pourvues de la propriete daccroitre lactivite enzymatique de la carboxypeptidase et que seul le groupe des substances tensioactives anioniques est depourvu de cette propriete.

Sur la dénaturation thermique et la dénaturation de surface de la carboxypeptidase a du pancréas. Influence du tween 80

Resume Thermic inactivation of carboxypeptidase A of the pancreas at temperatures ranging from 45° to 55° follows kinetics of the first order: k 50° = 1.52·10 −3 sec −1 . The activation energy, variation of enthalpy, variation of free energy, and variation of entropy, corresponding to this denaturation are, respectively: E = 49,600 cal/mol , Δ H ∗ = 49,000 ccal/mol , Δ F ∗ = 23,100 cal/mol , Δ S ∗ = + 81 cal/mol/degree . The presence of Tween 80 scarcely modifies these values. Inactivation through surface tension forces becomes very important in dilute solutions (I μg/ml). It first becomes manifest at a temperature of 20°; it is very sensitive, especially to the shape of the vessel in which the solutions are contained and is provoked by simple dilution. Presence of Tween 80 retards this inactivation considerably.