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Biomedical subjects

F Le Goffic

Publications and source records attributed to F Le Goffic.

At least 55 records · Page 3Linked to original sources
PubMed1984

[Photochemical affinity labeling of the macrolide binding site on the 70S E. coli ribosome].

Photoactivation of the alpha, beta-unsaturated ketone-epoxide system of [3H] dihydrorosaramycin at a wavelength above 300 nm allows the covalent attachment of the antibiotic to its receptor site. The radioactivity is mainly associated to proteins L1, L5, L6, S1; as a consequence, the binding site of this type of drug could be located at the peptidyltransferase center and in between both subunits.

Affinity Labels↗
PubMed1983

Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationship between the two activities.

In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated.

Acetyltransferases↗
PubMed1983

Photo-induced labelling of Escherichia coli ribosomes by a tobramycin analog.

An [3H]azidobenzyl derivative of tobramycin, a 4,6-disubstituted 2-deoxystreptamine aminoglycoside, has been synthesized, and its ability to label Escherichia coli 70-S ribosomes under photoactivation has been studied. Two concentrations of the photolabel, corresponding to the saturation of the two classes of tobramycin sites on the ribosomes, were used. The results show that, at high antibiotic concentrations which induce maximal misreading during protein synthesis, most of the ribosomal proteins are labelled. At low antibiotic concentration, which results in the saturation of the first-class sites, a few proteins of both subunits are labelled, including L6, S4, S5, and, to a lesser extent, L2, L13 and S18. The 30-S subunit is, on the whole, labelled more efficiently than the 50-S subunit.

Affinity Labels↗
PubMed1983

Photoaffinity inhibition of peptide transport in yeast.

A photoaffinity label, 4-azidobenzoyltrimethionine has been synthesized. It competitively inhibits trimethionine uptake in the yeast C. albicans. Upon UV irradiation it irreversibly and specifically blocks oligopeptide uptake. These results give the first example of photoinhibition of peptide uptake in yeast.

Affinity Labels↗
PubMed1982

Effects of N-alkylation and n-acylation on tobramycin activity.

The activities of tobramycin derivatives acetylated and ethylated on the 6'-N,2'-N and 3-N positions were examined. The MICs of these derivatives against tobramycin sensitive strains indicated that 2'-N-ethylated and 6'-N-ethylated derivatives have a fairly good activity, and confirmed that the 3-N position is the most important one for antibiotic activity since 3-N derivatives were less active. The MICs of these derivatives against tobramycin resistant strains, and their inactivation by tobramycin modifying enzymes were examined. These results showed that 2'-N or 6'-N ethylation protects the drug against inactivation by AAC(2') or AAC(6'), respectively, and 2'-N-ethyltobramycin and 6'-N-ethyltobramycin were active against strains containing these modifying enzymes. On the other hand, 3-N ethylation protects the drug against inactivation by AAC(3) but 3-N-ethyl tobramycin does not inhibit strains containing this enzyme.

Acylation↗
PubMed1982

Mechanism of action of a 16-membered macrolide. Characteristics of dihydrorosaramicin binding to Escherichia coli ribosome and the effects of some competitors.

The macrolide [3H]dihydrorosaramicin binds specifically to 50S and 70S bacterial ribosomal particles. We have studied the influence of salts, pH and additives on the interaction and found that the optimum requirement for salts was 10 mM trs-HCl (pH 7.6), 6 mM MgCl2, 60 mM NH4Cl, and that beta-mercaptoethanol which reacts on rosaramicin and its dihydro derivative cannot be used. The parameters of the binding were not dependent on the technique used, i.e. equilibrium dialysis, ethanol precipitation or two-phase partitioning. In our search for effectors of this binding, we have found that it is inhibited by other macrolides, little effected by tobramycin and chloramphenicol and enhanced by puromycin.

Anti-Bacterial Agents↗
PubMed1982

[Hydrosoluble polymers of NAD+ and ADP. Study of their coenzymatic properties as a function of their size].

Hydrosoluble polymers of NAD+ and ADP were synthesized according to a described method with some modifications. The cofactor was bound to the matrix by a spacer group of five atoms at the exocyclic adenine C-6 amino group. Cofactor incorporations were very high. The loading of NAD+ polymers were: 420 mumol NAD+/g polymer and 330 mumol ADP ribose/g polymer arising from degradation of NAD+; the loading of ADP polymers were: 1.40 and 1.43 mmol ADP/g polymer. Each polymer obtained in a same polymerization step was fractionated in two parts by gel filtration in such a manner that these two parts had the same framework but different molecular weights. The biological properties of the fractionated polymers were compared. The small sized polymers of NAD+, assayed with four dehydrogenases, were more reduced enzymatically than the larger ones and had the higher reduction rates relative to free NAD+. In a coupled system (L-lactate dehydrogenase, formate dehydrogenase) with coenzyme regeneration, the rate of production of L-lactate at the steady state of the system using NAD+ polymers of small size was 80% that of the system using free NAD+. The small-sized polymers of ADP were more phosphorylated by pyruvate kinase and creatine kinase than the larger ones. But the relative rates of transformation by pyruvate kinase of large sized polymers were higher than those of smaller ones.

Adenosine Diphosphate↗
PubMed1980

Photo-induced affinity labeling of Escherichia coli ribosomes by chloramphenicol.

In order to obtain more information about the binding site for chloramphenicol (D-threo diastereoisomer) on the bacterial ribosome, photo-affinity labeling experiments of this receptor have been performed with [3H]chloramphenicol itself. Control experiments show that this drug can be split photochemically by ultraviolet irradiation, whereas the ribosome is not modified structurally or functionally by such a treatment. When photolysis of a mixture of chloramphenicol and ribosomes is performed under critical conditions, some proteins like L1, L11, S3 and S4 are radiolabeled. L11, S3 and S4 are radiolabeled specifically as demonstrated by photo-incorporation experiments with isotopically diluted [3H]chloramphenicol or by comparison of the results obtained here with reversible experiments performed by the isotopic dilution method. When the D-erythro diastereoisomer of chloramphenicol is photo-incorporated into the bacterial ribosome, proteins are radiolabeled only in a non-specific way. These results show that this material could be used as an efficient scavenger. When finally D-threo [3H]chloramphenicol is photo-incorporated in the presence of a large amount of the D-erythro diastereoisomer, the radiolabeling pattern obtained for the proteins is quite different from that expected: while L11 is still labeled fairly extensively, L27 is the most radiolabeled protein found.

Affinity Labels↗
PubMed1980

A two-step synthesis of new water-soluble polymers of NAD+ and ADP. The biological properties of these polymers.

Alkylation at the N-1 position of the adenine moiety of NAD+, ADP or ATP with 2,3-epoxypropyl acrylate, followed by polymerization with or without acrylamide at pH 8, gave water-soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C-6 amino group. Cofactor incorporations were good to high: 145-447 mumol NAD+/g polymer and 667 mumol ADP/g polymer. About 30% of the bound NAD+ could be reduced with rabbit muscle lactae dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84% of the bound ADP was phosphorylated with rabbit muscle creatine kinase. High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35-81% that of free NAD+. These values remained substantially high with agarose-immobilized alcohol dehydrogenase (15-36%) and should eventually allow their use in continuous enzymatic reactors. Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 mumol ATP/g polymer were transformed with yeast hexokinase.

Acrylic Resins↗
PubMed1980

Have deoxystreptamine aminoglycoside antibiotics the same binding site on bacterial ribosomes?

(3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.

Aminoglycosides↗
PubMed1979

Mechanism of action of aminoglycoside antibiotics. Binding studies of tobramycin and its 6'-N-acetyl derivative to the bacterial ribosome and its subunits.

6'-N-[14C]Acetyl-tobramycin and [3H]tobramycin were synthesized and their binding to Escherichia coli ribosomes and ribosomal subunits studied using equilibrium dialysis. THE 70-S ribosome, as well as its 50-S and 30-S subunits, bound tightly to 6'-N-acetyl-tobramycin. The binding of [3H]tobramycin to ribosomes was quite different. The 70-S ribosome was observed to possess several classes of binding sites; of these, one was determined to be of higher affinity and lower capacity, the 6'-N-[14C]acetyl-tobramycin site. The isotopic dilution method was used to define the specificity of the interaction. The selective binding of 6'-N-[14C]acetyl-tobramycin was highly reversible by tobramycin, kanamycins A, B, C and neomycin, but not by streptomycin or erythromycin. Gentamicin C1a was a poor inhibitor. This suggested that either the kanosamin or garosamin rings might be determinant in the binding of these molecules, as well as the 6'-amino group.

Anti-Bacterial Agents↗
PubMed1979

Binding of tobramycin to Escherichia coli ribosomes: characteristics and equilibrium of the reaction.

A sample of [3H] tobramycin (5,000 Ci/Mole) has been synthetized and incubated with the bacterial ribosome and its subunits. The results obtained show that this antibiotic has two types of binding sites. The primary one is probably responsible for the inhibition of protein synthesis whereas the secondary one is probably related to the misreading and reading through of the messenger RNA.

Anti-Bacterial Agents↗