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Y Boulanger

Publications and source records attributed to Y Boulanger.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structural effects of the selective reduction of amide carbonyl groups in motilin 1-12 as determined by nuclear magnetic resonance.

Motilin is a 22-residue peptide stimulating stomach and intestinal motility. The motilin 1-12 fragment displays biological effects similar to the native peptide. Selective reduction of the amide carbonyl groups to form CH2NH analogs leads to a significant reduction in activity for the first two N-terminal positions and to a complete loss of activity for all other positions. The structures of motilin 1-12 and ten reduced analogs were investigated using the temperature dependence of the amide NH chemical shifts. In all the analogs, the structure of the N-terminal region (residues 1-5) was different from the structure of motilin 1-12, which is characterized by hydrogen bonding between Phe1 and Ile4. The structure of the C-terminal region of analogs was similar to the structure of motilin 1-12 for the first two reduction positions only (1-2 and 2-3), indicating that the C-terminal portion of motilin 1-12 is more critical for biological activity. Complete structural characterizations of motilin 1-12, [CH2NH]1-2, and [CH2NH]4-5-motilin 1-12 were performed by two-dimensional NMR spectroscopy and molecular modeling. The structural features observed confirm the differences based on the temperature dependence of the amide NH chemical shifts. These results demonstrate that conservation of the amide bond rigidity is essential for the activity of non-hydrolyzable analogs.

Amides↗
PubMed1995

Could cytoplasmic concentration gradients for sodium and ATP exist in intact renal cells?

In renal cells, the Na+ pump maintains a transmembrane concentration gradient for sodium ensuring the net reabsorption of sodium with or without cotransported species. This process requires a significant fraction of the ATP turnover of proximal tubules and thick ascending limbs. To understand the potential regulatory influences of Na+ and ATP on the activity of the Na+ pump in these nephron segments, the apparent kinetics of the membrane-bound Na+-K+ ATPase and of the cellular Na+ pump were studied in different preparations of dog proximal tubules and thick ascending limbs (tubular suspensions, tissue homogenates, and basolateral membrane vesicles) obtained from dog kidney cortex and red medulla. Two determinant kinetic parameters, i.e., the apparent Michaelis constant (Km) and the saturating concentrations for sodium and ATP, were compared with the intracellular concentrations of Na+ and ATP measured under physiological conditions. In both types of tubules, the apparent Km value for Na+ (5-15 mM) is set well below the measured mean intracellular concentration of sodium (50-60 mM), suggesting that the Na+ pump should be saturated by sodium ions under normal conditions. Nevertheless, a modest increment of the Na concentration in the vicinity of the pump, obtained by equilibrating the intra- and extra-cellular sodium concentrations at various extracellular [Na+] with nystatin, increases the activity of the Na+ pump in intact cortical tubules and thick ascending limbs, even when the extracellular [Na+] is set at the estimated intracellular [Na+], demonstrating that the pump is not saturated by sodium in situ. Similarly, the kinetics of the renal Na+ pump as a function of the ATP concentration suggested that the pump should be saturated by ATP in physiological conditions, since in both tissues the cellular ATP level (3-6 mM) is higher than the concentration required to achieve saturation of this activity (< 2.5 mM). However, in renal cortical tubules, the steady-state intracellular [Na+] is affected by modest changes of ATP concentration, suggesting that the Na+ pump is not functionally saturated by ATP. Our data suggest that concentration gradients for Na+ and ATP may exist in the cytosol of renal cells. These gradients would be related to the polarity of sodium transport and of the ATP-consuming and ATP-regenerating processes in intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1995

Structure-activity studies of the C-terminal segment of structurally reduced analogues of ET-1.

Structurally reduced analogues of endothelin-1 (ET-1) were synthesized by linking via aminocaproic acid (Aca) the segment 3-11 of ET-1 to C-terminal fragments of various lengths [16-21, 17-21,...,21]. Analogues were studied in their linear or cyclic form in the absence or presence of a formyl group on the Trp21 side-chain, and their biologic activities were tested using guinea pig lung parenchymal strips. The absence of the first disulfide bridge and the presence of the Aca spacer caused a slight decrease in activity. The presence of His16 is essential for the contractile activity of the monocyclic peptides. Formylation of these monocyclic analogues did not modify this behavior. Similarly, linear analogues carrying S-acetamidomethyl (Acm) functions and an Aca linker were still active. However, most formylated linear derivatives were partial agonists, whereas the addition of His16 caused a decrease in contractile activity. In these latter analogues, molecular modeling studies suggested that formylation produces different conformations.

Animals↗
PubMed1991

Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase.

Phenylalanyl-tRNA synthetases [L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20] from Escherichia coli, yeast cytoplasm, and mammalian cytoplasm have an unusual conserved alpha 2 beta 2 quaternary structure that is shared by only one other aminoacyl-tRNA synthetase. Both subunits are required for activity. We show here that a single mitochondrial polypeptide from Saccharomyces cerevisiae is an active phenylalanyl-tRNA synthetase. This protein (the MSF1 gene product) is active as a monomer. It has all three characteristic sequence motifs of the class II aminoacyl-tRNA synthetases, and its activity may result from the recruitment of additional sequences into an alpha-subunit-like structure.

Amino Acid Sequence↗
PubMed1991

Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.

Amino Acid Sequence↗
PubMed1991

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases.

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].

Amino Acid Sequence↗
PubMed1991

Structural determination of the vasoactive intestinal peptide by two-dimensional H-NMR spectroscopy.

The structure of the vasoactive intestinal peptide 1-28 in 40% 2,2,2-trifluoroethanol was investigated by two-dimensional 1H-nmr spectroscopy. All 1H resonances, except the gamma, delta, and epsilon protons of the lysine residues, could be sequentially assigned. Numerous intraresidual as well as short-range interresidual nuclear Overhauser effect spectroscopy connectivities were observed. Using a variable-target function minimization, a molecular model consisting of two helical stretches involving residues 7-15 and 19-27 connected by a region of undefined structure was calculated. The existence of an undefined structure between residues 16 and 18 confers mobility to the peptide molecule.

Amino Acid Sequence↗
PubMed1991

The complete amino acid sequence of bovine antithrombin (ATIII).

Bovine antithrombin (ATIII) is a glycoprotein of Mr 56,600. Its primary structure was established using peptide sequences from five different digests. Bovine ATIII exhibits four glcosylation sites as well as human ATIII. The primary structures of bovine and human ATIII were compared: all the residues required for the integrity of the heparin-binding domain are strictly conserved. However, there are differences in the secondary structures of both proteins, bovine and human ATIII.

Alkylation↗
PubMed1991

Effects of isoflurane, halothane, and enflurane on myocardial flow and energy stores in the perfused rat heart.

The effect of three volatile anesthetics (halothane, enflurane, and isoflurane) on coronary flow and metabolic state of isolated rat hearts was studied. These anesthetics are coronary dilators and their effects are dose dependent. At 2 MAC (minimum alveolar concentration), isoflurane, enflurane, and halothane increase coronary flow by 114 +/- 5.9, 93 +/- 6.1, and 77 +/- 6.4%, respectively (p less than 0.001). At these concentrations, they also have a modest but significant metabolic effect causing a 30% reduction in myocardial ATP and phosphocreatine levels, with no significant modification in ADP and AMP concentrations. Energy charge and lactate/pyruvate ratio were also unaffected by these anesthetics. The vascular and metabolic effects were reversible within 2 and 30 min, respectively. Perfusion of the hearts with a Krebs-Henseleit solution without Pi did not interfere with the vascular and the metabolic effect of the anesthetics; however, in this case, ATP and phosphocreatine concentration did not return to control levels after their discontinuation despite full recovery of the vascular effect. These data suggest that the volatile anesthetics have direct coronary vascular and myocardial metabolic effects and that these effects occur independently.

Adenosine Diphosphate↗
PubMed1991

Basolateral glucose transport in distal segments of the dog nephron.

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.

Aerobiosis↗
PubMed1991

Metabolism of lactate by a suspension of dog thick ascending limbs: relations with transport.

The addition of substrate in the form of lactate (L), but not glucose (G), increases the respiration of canine thick ascending limb (TAL) segments in a saturable (above 2 mM) fashion. More than 60% of this stimulation is ouabain-sensitive (1 mM ouabain) even if L and G transport are both sodium-insensitive processes in TAL. Thus L, but not G, specifically stimulates Na+ entry in TAL cells and its subsequent transport by the Na+,K(+)-ATPase. If chloride is substituted for by gluconate, no significant substrate-induced stimulation of ouabain-sensitive respiration is observed. SITS (4-acetamino-4'-isothiocyanostilbene-2,2'-disulfonic acid) also interferes with the L-induced stimulation of respiration. Thus L entry in TAL appears to be directly or indirectly coupled to the transepithelial flux of Cl-. Furosemide (F), but not amiloride, also inhibits this stimulation suggesting that the accelerated Na+ entry triggered by the application of L occurs through the F-sensitive carrier or that lactate transport is F-sensitive in TAL cells. In accord, F specifically impairs the metabolism of L (as compared to G). These data suggest that in intact TAL tubules both lactate uptake and oxidation are directly or indirectly influenced by the transcellular flux of NaCl. This organization may participate to maintain a stoichiometry between the transport of NaCl and the availability of L to support the energetic needs of TAL cells.

Animals↗
PubMed1990

The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity.

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.

Adenosine Triphosphate↗
PubMed1990

NMR visibility of 39K and 35Cl in erythrocytes and kidney tubules.

The NMR visibility of 39K and 35Cl has been investigated in erythrocytes and in dog renal tubules. In erythrocytes, the 39K NMR visibility was determined by comparing the signal intensities before and after hemolysis with water and by comparing the NMR and flame photometry results. Both procedures showed a NMR visibility of 100% for intracellular potassium. The visibility of intracellular chloride in erythrocytes was estimated at 40% by monitoring the intensity of the 35Cl signal as a function of the hematocrit value. In the case of kidney proximal tubules, the 39K visibility appeared to be very low but could not be accurately determined due to the low sensitivity of the nucleus. The 35Cl signals for intracellular chloride in renal tubules were too broad to be detected.

Animals↗
PubMed1990

Variable NMR visibility of intracellular sodium induced by Na(+)-substrate cotransport in dog cortical tubules.

The intracellular sodium concentration [( Na+]i) of dog kidney cortical tubules was monitored by flame photometry and 23Na NMR using dysprosium tripolyphosphate as shift reagent. Upon addition of substrates cotransported with sodium, flame photometry showed an increase in [Na+]i while no change (glutamine, glucose) or even a decrease (lactate) in the Na+i NMR signal was observed. This discrepancy could not be explained by a lack of ATP prior to the addition of substrates or by a decrease of NMR visibility of Nai+ induced by binding of substrate to membrane transporters (and pump). We propose that a variation of the "apparent visibility" of Nai+ may occur, arising from either a compartmentation of Nai+ in dog cortical tubules or an inhomogeneous extracellular distribution of the shift reagent.

Adenosine Triphosphate↗
PubMed1990

Relationship between intracellular ATP and the sodium pump activity in dog renal tubules.

To examine the potential effect of the cellular ATP concentration and of the phosphate potential on the function of the sodium pump in intact renal cells, the ATP content of dog cortical tubules was first modified by a 30-min preincubation with one of the following effectors: 5 or 10 mM fructose, 2.5 mM adenosine 5'-monophosphate (AMP), or 2.5 mM adenosine in the presence of substrates (10 mM glutamine + 1 mM glutamate with either 10 mM lactate (low ATP) or 10 mM pyruvate (high ATP)). The tubules were then incubated in Krebs-Henseleit saline using two different phosphate concentrations and the same substrate mixture. The ATP content in tubular cells was modified by these treatments, ranging from 2.2 to 5.7 mM. The oxygen uptake by the tubules was measured before and after application of a small amount of nystatin (0.05 mM, 6 mumol/g wet wt.), added to impose an identical and submaximal increment of work to the Na(+)-K+ ATPase in tubules, irrespective of their ATP condition. This manoeuvre was followed by the addition of 1 mM ouabain to inhibit the sodium pump and quantify the respiration related to the activity of the Na+ pump. No significant effect of the ATP content on the respiratory cost of the Na(+)-K+ ATPase activity was noted when the [ATP] was above the normal concentration of approximately 3.0 mM before or after introduction of nystatin. In a second group of experiments, tubules were treated with 0.1 mM digitonin (13 mumol/g wet wt.) and resuspended in intracellular-like and sodium-free medium. The respiration was measured before and after the addition of increasing Mg-ATP concentrations (0-12 mM). A fixed quantity of Na+ (20 mM) was then introduced before ouabain was applied. The oxygen uptake was measured in these three conditions. We observed a fixed increment of ouabain-sensitive respiration upon stimulation of the pump activity by sodium at ATP concentrations ranging from 2 to 7 mM. The same observation applied when the free energy released from ATP hydrolysis ranged from -50 to -56 kJ.mol-1 and when the [ATP]/[ADP].[Pi] ratio ranged from 1.5 to 7.5 mM-1. These results suggest that the Na+:ATP stoichiometry of the Na(+)-K+ ATPase is not modified by [ATP] in dog cortical tubules when the ATP content is at or above the physiological value. Furthermore, the stoichiometry of the pump does not appear to change when the phosphate potential and (or) the free energy released from ATP hydrolysis are altered.

Adenosine Triphosphate↗
PubMed1989

Identification of the major tRNA(Phe) binding domain in the tetrameric structure of cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast.

Native cytoplasmic phenylalanyl-tRNA synthetase from baker's yeast is a tetramer of the alpha 2 beta 2 type. On mild tryptic cleavage it gives rise to a modified alpha 2 beta 2 form that has lost the tRNA(Phe) binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated beta subunit. Each purified peptide was unambiguously assigned to a unique stretch of the beta subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172-Ile 173 bond is the unique target of trypsin under mild conditions and that the N-terminal domain of each beta subunit (residues 1-172) contains the major tRNA(Phe) binding sites.

Amino Acid Sequence↗
PubMed1989

An incubation system for the NMR study of kidney tubules.

Isolated kidney cortical tubules require a very rapid oxygen supply and mechanical agitation to be optimally functional. A sample chamber in which a tubule suspension is oxygenated by recirculating oxygen gas inside a coil of dialysis fibers to avoid cell loss through bubbling and in which the tubules are agitated by a gas-driven turbine has been designed. In such a system, dog cortical tubules (35-45 mg/ml) were found to be metabolically stable for more than 3 h as indicated by linear lactate consumption and glucose production. Small pH variations resulting from carbon dioxide and bicarbonate productions were measured. Good-quality 23Na NMR spectra of dog kidney cortical tubules were recorded with such a system, allowing a 1-min time resolution.

Animals↗