S U M M A R YP-Lactamases (EC. 3 . 5 . 2 . 6 ) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of P-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate P-lactamase in mutants previously reported to lack the enzyme. R factor RTEM and RPI P-lactamases that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R I and RGNI1, with identical isoelectric focusing patterns, have the same biochemical properties. Chromosomal and R factor-mediated P-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing, R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of P-lactamases carried by R factors. I N T R O D U C T I O NIsoelectric focusing (Haglund, 1967) is a method of separation in which proteins align themselves as sharp bands at their isoelectric points (PI) in an electrophoretically-produced pH gradient. A high degree of resolution is obtained by the method, because focusing is caused by forces that act against diffusion and proteins are therefore concentrated during their separation. We have used the technique in the analytical mode for the separation of P-lactamases (EC. 3 . 5 . 2 . 6 ) on thin layers of polyacrylamide gel and have located the focused enzymes with a cephalosporin substrate which changes colour after degradation by P-lactamase. This technique of separation and specific staining of P-lactamases allows demonstration of low levels of activity and presents the different enzymes produced by various strains as patterns of bands that can easily be recognized and compared.The P-lactamases produced by Gram-negative bacteria are known to differ in their substrate specificities, inhibitor profiles, electrophoretic mobilities, biochemical properties and reactions with antisera ( Yamagishi et al. 1969). These enzymes have frequently been classified by the relative rates at which they hydrolyse penicillins and cephalosporins. Various methods have been used for estimating the activity of crude and pure enzyme preparations, and when a single strain produces two P-lactamases these have not always been separated before determining the substrate profile. Comparison of profiles determined in different laboratories may therefore be misleading. The use of isoelectric focusing of P-lactamases for direct comparison of different enzymes has been illustrated in this paper principally by reference to enzymes from Escherichia coli. This species was chosen because strains carrying R factors specifying well-characterized P-lactamases were available. METHODSStrains. These are listed in Table I. The clinical ...

The affinities of a range of penicillins and cephalosporins for ther penicillinbinding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant.

The competition of a number of beta-lactam morphogenic probes for the penicillin-binding proteins (PBPs) of Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, Proteus rettgeri, and Escherichia coli has been studied. The results indicate that the various gram-negative bacteria have similar, but not identical, PBP patterns and that the individual proteins probably perform similar morphogenic functions as in E. coli K-12. Comparison of the 50% binding concentrations of the compounds for the various PBPs of the five strains with their antibacterial activity indicates that the different antibiotics are excluded to a greater or lesser degree by the outer membrane permeability barrier and that the exclusion is most pronounced in P. aeruginosa.

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Actinic Prurigo (AP), an uncommon idiopathic photodermatosis, presents a distinct clinical picture and can be severely debilitating. The clinical features, investigation and treatment of AP are reviewed. We report the experience of an Australian photobiology unit with this condition.

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