Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028.

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.

Use of chromatofocusing for separation of beta-lactamases. VIII. Analytical chromatofocusing of chromosomal cephalosporinases from four Klebsiella strains.

Although still there are Klebsiella strains which do not harbour plasmids and produce constitutive chromosomal beta-lactamases, recently clinical isolates were found in ever increasing numbers carrying mainly TEM-, CARB- and OXA type R-factors. We selected four chromosomal cephalosporinase producing Klebsiella strains to study the pI values of the enzymes and their simultaneous separability from accompanying proteins by chromatofocusing techniques. We compared pI values of the pure and the crude preparations: K. pneumoniae K1 SC 10436: pIpure = 6.4, pIcrude = 6.42; K. aerogenes K1 1082 E: pIpure = 6.5, pIcrude = 6.5; K. oxytoca 1082 E: pIpure = 6.42, pIcrude = 6.4; K. oxytoca 20: pIpure = 7.62, pIcrude = 7.6. Excellent agreement of the pI values among each other, but occasional differences with those obtained by analytical isoelectrofocusing are attributed to methodological diversities and to the presence of satellite enzymes, known to exist in Klebsiella.

Purification of Escherichia coli B-specific p-aminobenzoate "pick-up" protein to homogeneity by affinity chromatography.

Of the satellite fractions of Escherichia coli B dihydrofolate synthetases, a non-enzymic protein that is specifically able to bind p-aminobenzoate and sulphonamides has been purified 6000-fold by p-aminobenzoylcellulose affinity chromatography. The protein was named p-aminobenzoate "pick-up" protein according to its function, i.e., to bring p-aminobenzoate into reaction with L-glutamate and pteridine during dihydrofolate biosynthesis. About 4 mg of pure protein (0.532% recovery, calculated from the total p-aminobenzoate binding capacity of the unfractionated supernatant separated from the crude bacterium plasma) can be obtained from 500 g of harvested cells. The product is homogeneous in polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecyl sulphate, and has a molecular weight of 15,000 daltons +/- 5% as measured by sodium dodecyl sulphate gel electrophoresis and Sephadex G-75 gel column chromatography. p-Aminobenzoate and sulphonamide ligand binding studies showed a single binding site per p-aminobenzoate pick-up protein molecule. KD values for p-aminobenzoate and some sulphonamides as well as for L-glutamate, L-gamma-glutamyl oligopeptides, some pteridines and folate antagonists are also presented in order to illustrate the specificity of the receptor protein.