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Publications (33)
Covalent embedding of a (eta(6)-arene) ruthenium(II) complex into the protein papain gives rise to a metalloenzyme displaying a catalytic efficiency for a Lewis acid-mediated catalysed Diels-Alder reaction enhanced by two orders of magnitude in water.
Recently we have found that the metallocarbonyl complexes (eta(5)-C(5)H(5))M(CO)(x)(eta(1)-N-maleimidato) (M=Fe, Mo, W; x=2 or 3) bearing a maleimide function were irreversible inhibitors of the enzyme papain. To get further insight into the binding mechanism of these compounds we synthesized the related complexes (eta(5)-C(5)H(5))M(CO)(x)(eta(1)-N...
The cationic (η6-arene)ruthenium complexes 6–9 containing a chloroacetamide or a maleimide functional group on the arene ligand were synthesized and successfully used to introduce ruthenium(II) species to the active site of the cysteine endoproteinase papain in a site-directed and covalent fashion as shown by enzymatic and ESI-MS studies.
An investigation on the presence of mycotoxin patulin was performed on 71 apple juices and concentrates and 21 infant fruit purees purchased from retail outlets or producers in Tunisia, by reversed-phase high-performance liquid chromatography using a C18 column with UV detection at 276 nm, keeping the recovery higher than 96% for spiking levels ran...
Site-directed and covalent introduction of various transition metal-organic entities to the active site of the cysteine endoproteinase, papain, was achieved by treatment of this enzyme with a series of organometallic maleimide derivatives specially designed for the purpose. Kinetic studies made it clear that time-dependent irreversible inactivation...
Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody-antigen binding...
Patulin is a mycotoxin produced by fungal species that frequently grow on fruit and vegetables. It presents risks, particularly for children consuming compotes and fruit juices. Thus, it is important to have methods such as immunoassays to screen a large number of samples. In the relevant literature, previous studies on the production of antibodies...
A plasma process using NH3 was developed in order to incorporate amine functions on the surface of nitrocellulose membranes for an oriented covalent attachment of antibodies through their oxidized carbohydrate moieties. An average covalent binding capacity of 60 μg/cm2 of antibodies was reached. The potential use of this process in developing envir...
The non-uniform affinity of polyclonal antibodies is a major problem when these antibodies are used in immunochemical-based environmental applications. A specific affinity chromatography procedure was developed to extract selectively anti-isoproturon antibodies from a crude polyclonal antiserum. These antibodies were fully characterized and helped...
Detectors with covalently immobilized bioactive compounds able to interact with selected analytes are needed for various kinds of biosensors. Protein macromolecules can be immobilized covalently on surfaces with appropriate functional groups such as amine groups. Low pressure plasmas of NH3 and NH3/H2 mixtures were used to incorporate a maximum of...
The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42+/-0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirm...
A one‐step technique for the purification of isoproturon antibody has been developed to overcome the common elution problems encountered when high‐affinity polyclonal antibodies are used in immunoaffinity columns. Because of the non‐uniform affinity, desorption of the analyte can be problematic. Two herbicide derivatives were coupled to Sepharose C...
The controlled attachment of antibodies is a prime requirement for developing membrane-based immunoassays used in field tests. A novel dry process was developed to functionalize a nitrocellulose membrane by introducing amino groups on the surface, allowing an oriented covalent linkage of antibodies through their oxidized carbohydrate moieties. A no...
A gene library of the Leptospira meyeri serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a Leptospira DNA fragment carrying metY complemented Escherichia coli strains carrying mutations in metB. The latter gene encodes cystathionine gamma-synthase, an enzym...
A common problem in developing immunochemical-based analytical methods is that the desired antibodies represent only a small portion of the heterogeneous antibody population. Affinity purification of polyclonal serum requires the correct choice of ligands used to extract antibodies featuring similar characteristics in their recognition properties (...
An indirect enzyme immunoassay (EIA) for the detection of the phenylurea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.64 ng/mL. The sensitivities were 0.07 ng/mL (IC80) and 0.02 ng...
Escherichia coli dihydroorotase contains six cysteines/subunit, which are potential ligands of structural and catalytic zinc metals at protein sites of the enzyme. Specific thiol reagents modify, in nondenaturing conditions only, two of these cysteines; these two residues are thought to be ligands of structural zinc. We report here on the localizat...
The Leptospira meyeri serovar semaranga metX gene was identified by complementation of an Escherichia coli metA mutant, i.e., devoid of homoserine O-succinyltransferase. However, the MetX protein exhibited a homoserine O-acetyltransferase activity in agreement with its similarity to homoserine O-acetyltransferases. Reverse transcription-PCR analysi...
A reasonably facile and effective procedure is described for preparing the optically active substrate of dihydroorotase (EC 3.5.2.3), N-carbamyl-L-aspartate (L-CA), which is not commercially available. Compared to the previously described methods, this procedure is not plagued by side reactions, and the L-CA is obtained in a solid form as the Mg2+/...
The development of new immunosorbents for the selective solid-phase extraction of phenylurea and triazine herbicides from environmental waters is presented. The production and purification of polyclonal antibodies against isoproturon and atrazine are described. The immunoconjugates were characterized by electrospray mass spectrometry before immuniz...
The phosphopyridoxyl peptides of beta cystathionase and cystathionine gamma synthase of Escherichia Coli were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver gamma cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 21 3790-3794.) showed...
The phosphopyridoxyl peptides of β cystathionase and cystathionine γ synthase of were identified after reduction, carboxymethylation and proteolysis of the holoenzymes. Their comparison with those obtained from rat liver γ cystathionase (Fearon, C.W., Rodkey, J.A. and Abeles R.H. 1982. Biochemistry 3790 – 3794.) showed a high degree of homology bet...
A homology has been observed between aminoacid sequences of the PLP binding sites of the two enzymes of cystathionine metabolism.
Cystathionine-γ-synthase and β-cystathionase from Escherichia coli are inhibited by aminoethoxyvinylglycine in similar time-dependent and reversible processes.
The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of beta-cystathionase, the MetC gene product. The metC gene (1185 nucleotides) encodes a protein having 395 amino acid residues. T...
The allosteric phosphofructokinase from Escherichia coli has been renatured after complete unfolding in concentrated guanidine hydrochloride. The enzyme regains both its catalytic and regulatory abilities quantitatively. The kinetics of reactivation are biphasic and are consistent with a two-step mechanism in which a monomolecular reaction precedes...
In oligomeric proteins, the native conformation and its functional properties depend on the interactions which exist between the different chains. The role of these subunit interactions can be studied using either the unfolded state or the native state as a starting point. During the folding process, the properties which appear following a bimolecu...
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-aminoglycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification proce...
A wild-type strain of Staphylococcus aureus that inactivates the 4,6-glycosidically linked deoxystreptamine aminoglycoside antibiotics by a plasmid-mediated process
was found to harbor two enzymes: an acetyltransferase of the AAC(6′) type and a new phosphotranferase specific to the gentamicin
components. The target of this last enzyme is the 2″-hyd...
A wild-type strain of Staphylococcus aureus, which inactivates a wide variety of aminoglycosides (except the gentamicin components), has been found to harbor a plasmid
(RAp01) that mediates the biosynthesis of a nucleotidyltransferase. This enzyme modifies the 4′-hydroxy function of these
antibiotics. The plasmid has been studied, the enzyme respon...
A new R factor-mediated gentamicin acetyltransferase obtained from a clinical isolate of gentamicin-, tobramycin-, and kanamycin-resistant
Klebsiella was characterized. R factor R 176 was transferred to Escherichia coli K-12 LA 290 from a Klebsiella. Resistance transfer, minimal inhibitory concentrations of various aminoglycoside antibiotics, enzym...
Des Moraxella résistantes à la kanamycine, au BBK 8 et à la tobramycine biosynthétisent une acétyltransférase qui acétyle les antibiotiques précédents sur l'azote 6′. Cette protéine est une isoenzyme de la kanamycine acétyltransférase.
