Biomedical subjects
Publications and source records attributed to F Le Goffic.
Our purpose being to work out a "continuous flow" enzymatic reactor of acetylation with a cofactor, we present here some results about the immobilization of coenzyme A on T40 dextran. Two methods of fixation are compared with regard to the capacity and the biological activity of the polymers thus obtained.
In order to produce specifically N-monoalkylated derivatives of aminoglycoside antibiotics of potential therapeutic values, we have developed an enzymatic reactor. This system uses the aminoglycoside acetyltransferase as catalyst and acetylcoenzyme A as acetyl donor. The immobilization of one aminoglycoside acetyltransferase on different resins has been studied. The coreticulation of this enzyme on DEAE cellulose in the presence of glutaraldehyde gives rise to an enzymatic resin of high efficiency. On the other hand, we have also studied the acetylation of coenzyme A in a simple manner. Acetylation occurs in a quantitative yield when the reaction is performed in the presence of polyvinyl-4 pyridine/divinylbenzene 2 per cent. These conclusions enabled to develop two types of acetylating reactors which give rise without purification to 3-acetyl gentamicin.
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Gram-negative bacilli isolated from clinical specimens submitted for culture in two Paris hospitals during 1974 were studied for susceptibility to six currently used aminoglycosides: kanamycin, neomycin, paromomycin, lividomycin, gentamicin, and tobramycin. Resistance patterns of strains of various species including those of Enterobacteriaceae, Pseudomonas, and Moraxella were determined, and the strains were grouped into eight resistance "phenotypes." In comparative studies of 807 strains belonging to different phenotypes, amikacin was markedly more active than any of the six other antibiotics; at concentrations of less than or equal to 4 mug/ml, it inhibited about 88% of the strains, including those resistant to gentamicin and tobramycin. Some amikacin-resistant strains were found among different species. The mechanism of resistance to amikacin of strains of Serratia and Moraxella group II was related to an N-acetylating enzyme. Amikacin can be expected to be useful as an alternative treatment of infections due to gram-negative bacilli sensitive to aminoglycosides and also, more particularly, for the treatment of patients infected with multiresistant strains.
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 responsible for this resistance pattern has been isolated by affinity chromatography, and its kinetics and physicochemistry have been characterized. The target of this enzyme has also been located by demonstrating the structure of one inactivated compound, 4'-(O)-adenylyltobramycin.
A new nucleotidyltransferase has been obtained from clinical isolates of kanamycin, amikacin, tobramycin, neomycin and lividomycin resistant strains of Staphylococcus aureus. The enzyme has been purified by affinity chromatography then characterized in a kinetic and physicochemical point of view. The results obtained support that the target of this enzyme is the 4'-hydroxyl group of a number of aminoglycosides. We propose to call it ANT(4')I.
On-line computerized treatment of enzyme kinetic data allows the precise measurement of Michaelis--Menten constants (Km and V) from a single progress curve. This method has been used to determine the kinetic constants of a beta-lactamase extracted from an Escherichia coli strain. In the profile of enzymatic activity there obtained, Km and V are a function of the pH. From these results some information is derived about the mechanism of the enzyme--substrate binding.
Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds. The enzyme is eluted with a gradient of sodium chloride or released with the substrate. This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.
This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.
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A beta-lactamase was extracted from an Escherichia coli K-12 strain carrying the R-TEM plasmid and has been purified by affinity chromatography. Antisera to this enzyme were prepared in the rabbit, and the enzyme-antibody neutralization reaction has been evaluated with acidimetric methods (pH stat or pH meter). Under defined experimental conditions, it is now possible to clearly illustrate the enzyme-antibody reaction by means of accurate, rapid, and simple-to-perform methods. These methods are in accord with the specificity of the two reactive partners and allow the detection of the enzyme in a crude bacterial extract which would eventually contain more than one beta-lactamase.
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, enzymatic inactivation, and the isoelectric point of the enzyme obtained were determined. We found that R factor R 176 could govern the enzymatic 3-N acetylation of gentamicin, tobramycin, and kanamycin, and isoelectric point determinations suggested that the enzymes governed by R 176 and R 135 are different. The genes responsible for resistance to gentamicin, tobramycin, and kanamycin govern the synthesis of an isoenzyme of AAC(3)I, which we propose to call AAC(3)II.
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