Étude thermodynamique de la transition hélice–pelote statistique en solvants mixtes de différents esters de l'acide poly(L-glutamique)

Helix–coil transition of poly(γ-methyl-L -glutamate), poly(γ-ethyl-L -glutamate), and poly(γ-benzyl-L -glutamate) has been studied in mixed solvents by calorimetry, polarimetry, and viscometry. The experimental data have allowed the evaluation of solvation enthalpy Δh_b, equilibrium constant K for hydrogen bond formation between the active solvent component and CO and NH groups, and the cooperativity parameter σ. The conformational transition of polypeptides in solution in a mixed solvent containing enough active solvent to maintain the coiled conformation has been produced by dilution with the helix-supporting solvent for the measurements of enthalpy of transition Δh_s. The average value for Δh_s is 3550 ± 300 J/mol and is practically independent of the nature of the side chain for the dichloroacetic acid-ethylene dichloride solvent pair at 25°C. A noticeable concentration effect exists in the case of poly(γ-benzyl-L -glutamate). The helical conformation is less stable for poly(γ-ethyl-L -glutamate), and this is explained by a steric effect hindering the access of dichloroacetic acid to side chains. Constant K has been calculated using polarimetric data and also from values of Δh_s obtained at different temperatures using the Bixon and Lifson theory on the one hand and that of Sayama and coworkers on the other hand. Values of σ for poly(γ-ethyl-L -glutamate) have been calculated according to both theories mentioned, and the results show that the two sets of values are quite similar. The constant σ depends on the nature of the active solvent, on temperature, and on the binary-solvent composition. These conclusions are confirmed by viscometric results. Values of Δh_b calculated from constant K are 5230 J/mol when Bixon and Lifson theory is used and 5569 J/mol when the theory at Sayama and coworkers is used. In both cases the value for Δh_b is much lower than that of an intramolecular hydrogen bond. Experimental results suggest that the solvation mechanism would proceed in a manner so that mechanisms described in both theories are involved.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Specific Antisera Against the Catecholamines: L-3,4-Dihydroxyphenylalanine, Dopamine, Noradrenaline, and Octopamine Tested by an Enzyme-Linked Immunosorbent Assay

Antisera were raised against L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catecholamine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the nonconjugated compound was poorly recognized. The nonreduced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.     

Salmonella-typhimurium-specific difference in rate of intracellular killing by resident peritoneal macrophages from salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice

The aim of the present study was to determine whether the difference between the rate of intracellular killing of Salmonella typhimurium by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice also holds for other salmonellae and other bacteria species. After in vivo phagocytosis, the initial rate of in vitro intracellular killing of S. typhimurium phagetype 505, S. typhimurium phagetype 510, and S. typhimurium M206 by macrophages of CBA mice amounted always to approximately 1.7 times the value found for macrophages of C57BL/10 mice (p less than 0.001), indicating that the difference in killing efficiency between CBA and C57BL/10 macrophages holds for various strains of S. typhimurium. However, some other salmonella species, i.e., S. dublin and S. heidelberg, as well as E. coli 054 and 02K1+, Listeria monocytogenes EGD and L347, and Staphylococcus aureus were killed equally efficiently by macrophages of both mouse strains. These findings indicate that the difference between the rates of intracellular killing by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 does not hold for several other bacteria species and thus might be specific for S. typhimurium. Subsequent experiments showed that the in vivo proliferation of S. typhimurium 510 in the first 2 days after i.v. injection was 2.0-fold to 3.0-fold higher in the spleens and livers of C57BL/10 mice than in those of CBA mice, whereas the in vivo proliferation of S. dublin and S. heidelberg was between 1.0-fold to 1.4-fold higher in the C57BL/10 mice. These findings suggest that the differences between the rate of in vitro intracellular killing of salmonella by CBA and C57BL/10 macrophages are reflected in differences in the rate of in vivo proliferation of these microorganisms in CBA and C57BL/10 mice. To gain insight into the involvement of the oxidative metabolism of CBA and C57BL/10 macrophages in the difference in the rate of intracellular killing of S. typhimurium, the O2 consumption and H2O2 release by resident peritoneal macrophages was determined. The amplitudes of the respiratory burst and the release of H2O2 was identical in macrophages of the two mouse strains after triggering by either preopsonized heat-killed S. typhimurium or phorbol myristic acetate. These findings indicate that the mouse species-associated difference in the intracellular killing of S. typhimurium is not caused by a difference in the oxidative metabolism of CBA and C57BL/10 macrophages.     

l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Population Heterogeneity of Agrobacterium tumefaciens in Galls of Populus L. from a Single Nursery

This study focused on the natural crown gall infections occurring in a Leuce poplar nursery. Soil effects on crown gall frequency were detected, indicating that contamination was due to a resident Agrobacterium tumefaciens population, which was present before seedling plantation. The crown gall frequency on poplar progenies varied from 3 to 67%, indicating the feasibility of improvement in crown gall resistance. Of 129 tumor isolates, 128 were pathogenic. These isolates were of biotype 1 or 2. Biochemical, serological, and antibiotic resistance typing results concurred, indicating the presence of four biotype 1 and two biotype 2 resident subpopulations. No significant change was noticed in the relative proportions of subpopulations from one year to another. Pathogenic subpopulations both in vitro and in planta were susceptible to Kerr K84 (P. B. New and A. Kerr, J. Appl. Bacteriol. 90:172-179, 1972). In addition, no serological cross-reactions were found to occur between K84 and the pathogenic subpopulations.     

Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.     

Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.