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W V Shaw

Publications and source records attributed to W V Shaw.

At least 91 records · Page 5Linked to original sources

Chloramphenicol acetylation in Streptomyces.

Twenty-one strains of actinomycetes were screened for the presence of chloramphenicol acetyltransferase, the enzyme responsible for chloramphenicol resistance in many species of bacteria. Only five strains, belonging to three species, yielded mycelial lysates which catalysed the formation of chloramphenicol acetates in the presence of acetyl-coenzyme A: Streptomyces coelicolor Müller, S. acrimycini, and S. griseus. A mutant of S. acrimycini selected for an increase in resistance to chloramphenicol had a higher specific activity for chloramphenicol acetyltransferase than that found in the parental strain; the enzyme was not inducible in the mutant, the parental strain, or any other strain tested. Chloramphenicol was not acetylated by lysates of a strain of S. venezuelae, the organism known to produce it.

Acetyl Coenzyme A↗

Chloramphenicol: an enzymological microassay.

A new assay for chloramphenicol in biological fluids has been developed that offers sensitivity, specificity, precision, accuracy, economy, and ease of performance. The assay is based on the enzymological acetylation of chloramphenicol catalyzed by an R factor-mediated enzyme. [(14)C]acetyl coenzyme A serves as the donor of the labeled acetyl group, and the product, [(14)C]acetoxychloramphenicol, is separated from the labeled precursor by utilizing its preferential extraction into benzene. The product is then quantified by liquid scintillation counting. This assay measures chloramphenicol concentrations in both plasma and other biological specimens and in the presence of other antibiotics, hemolysis, or jaundice. Its rapidity and ease of performance are useful for clinical laboratories, and its sensitivity allows determinations on 10 mul of plasma.

Acetylation↗

Chloramphenicol acetyltransferases specified by fi minus R factors.

Chloramphenicol acetyltransferases specified by fi(-) (F fertility-noninhibiting) R factors were compared with those specified by the fi(+) R factors R1, 222, and R6-S. Three classes of enzyme were distinguished: class I was determined by group N fi(-) R factors and was indistinguishable from the enzyme determined by fi(+) R factors, class II was correlated with group W fi(-) R factors, and class III was determined by R387, an unclassified fi(-) R factor.

Acetyltransferases↗

Mechanism of chloramphenicol resistance in staphylococci: characterization and hybridization of variants of chloramphenicol acetyltransferase.

Fifty clinical isolates of chloramphenicol-resistant staphylococci from diverse sources were screened for the presence of chloramphenicol acetyltransferase (CAT) and were found to contain the inducible chloramphenicol-inactivating enzyme in each case. Polyacrylamide disc gel electrophoresis revealed four distinct types of CAT of which three were purified to a state of homogeneity. Each purified CAT preparation was shown to exist as a tetrameric protein with a native molecular weight of 80,000 and an identical subunit size of 20,000. All four staphylococcal types of CAT exhibited identical catalytic and immunological properties, but they possessed variable sensitivity to heat denaturation and to inhibition by mercuric ion. Each of the three purified variants of staphylococcal CAT was capable of undergoing reversible denaturation in 6 m guanidine hydrochloride. In vitro hybridization was successful between pairs of each of the three purified staphylococcal enzymes. Only one heteromeric (hybrid) species was observed in addition to the parental types rather than the three predicted from the known quaternary structure of staphylococcal CAT.

Acetyltransferases↗

Hybridization of variants of chloramphenicol acetyltransferase specified by fi + and fi - R factors.

An fi(-) type of R factor for transferable chloramphenicol resistance carries the structural gene for a novel species of chloramphenicol acetyltransferase (EC 2.3.1.99). The enzyme associated with the fi(-) plasmid is distinct from that described for fi(+) R factors. The fi(+)- and fi(-)-related variants of chloramphenicol acetyltransferase were hybridized in vivo and in vitro. Both techniques yielded only a single symmetrical heteromeric species rather than the three (A(3)B(1), A(2)B(2), and A(1)B(3)) hybrids predicted from the tetrameric (identical monomers) structure of chloramphenicol acetyltransferase.

Acetyltransferases↗

Mechanism of Chloramphenicol Resistance in Staphylococcus epidermidis.

The mechanism of chloramphenicol resistance in several multiple-resistant Staphylococcus epidermidis strains has been studied and shown to be due to the presence of the enzyme, chloramphenicol acetyltransferase. As with S. aureus, the inactivating enzyme in S. epidermidis appears to be the product of a structural gene on the chloramphenicol plasmid because resistance and enzyme activity are concurcurrently lost after growth in acridine orange or at elevated temperatures. The synthesis of chloramphenicol acetyltransferase in S. epidermidis has been compared with the function of a similar enzyme in chloramphenicol-resistant S. aureus with the conclusion that the kinetics of induction, products of the reaction, and general properties of the enzymes are identical. The chloramphenicol acetylating enzyme from S. epidermidis has been purified to a state of homogeneity and compared with the analogous purified S. aureus enzyme. Both purified preparations consist of native enzymes with molecular weights of 80,000, and evidence is presented that is consistent with their being made up of four identical subunits of 20,000 each. The two staphylococcal enzymes are identical with respect to pH optimum, apparent affinity (K(m)) for chloramphenicol, heat denaturation, and immunological reactivity, but they differ in electrophoretic mobility, chromatographic behavior, substrate specificity, and sensitivity to inhibition by mercuric ion.

Journal Article↗

Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus.

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to d-threo chloramphenicol which lacks a C(3) hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.

Acyltransferases↗

Characterization of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus.

Chloramphenicol-resistant strains of Staphylococcus aureus contain an inducible enzyme which inactivates chloramphenicol by acetylation in the presence of acetyl coenzyme A. The products of acetylation are chromatographically indistinguishable from those obtained with chloramphenicol-resistant Escherichia coli harboring an R factor. The kinetics of induction of chloramphenicol acetyltransferase are complicated by the inducer's effect on protein biosynthesis and its fate as chloramphenicol 3-acetate, which is not an inducer of the enzyme. The E. coli and S. aureus enzymes have been compared, with the conclusion that they are identical with respect to molecular weight (approximately 78,000) and pH optimum (7.8), but differ with respect to heat stability, substrate affinity, electrophoretic mobility, and immunological reactivity. Antiserum prepared against enzyme from E. coli contains precipitating antibody, which inactivates the E. coli enzyme, but neither precipitates nor neutralizes the activity of S. aureus enzyme.

Acyltransferases↗