Hiroshi Ogawara

Bacillus cereus β-lactamase: Reaction with N-bromosuccinimide and the properties of the product

The effect of N-bromosuccinimide on the enzymatic activity and the conformation of a Bacillus cereus beta-lactamase (penicillin amido-beta-lactamase EC 3.5.2.6) was studied. Incubation with 10 muM N-bromosuccinimide caused over 95% decrease of the enzymatic activity within 15 min. Spectrophotometric titration with N-bromosuccinimide showed that the reaction proceeded in two steps. The half-inactivated enzyme was prepared by the reaction with N-bromosuccinimide and its properties examined. Amino acid analysis showed that the half-inactivated enzyme contained one residue of tryptophan less while other amino acid contents were similar. Neither the molecular weight nor the mobility in disc electrophoresis was changed. However, the affinity to a cephalexin-CH-Sepharose column was increased, and the Km value for cloxacillin was one-third that of the native enzyme, although that for benzylpenicillin was similar. These results indicate that a tryptophan residue sensitive to N-bromosuccinimide is essential for the maintenance of the rigid conformation and that its oxidation alters the enzyme in a manner such that a substrate with a bulky group in its side chain can form an enzyme-substrate complex more easily. In the native enzyme, the value of (f(a))(eff) (Lehrer, S.S. (1971) Biochemistry 10, 3254-3263), did not vary significantly in the absence or the presence of cloxacillin. In contrast, in the half-inactivated enzyme the presence of cloxacillin affected the conformation such that over two thirds of the tryptophyl fluorescence were accessible for quenching by KI, although about half was accessible in the absence of cloxacillin.

Synthesis and chain length-anti-HIV activity relationship of fully N- and O-sulfated homooligomers of tyrosine.

Fully N- and O-sulfated homooligomers from octamer to nonadecamer of tyrosine were obtained as their sodium salts, aO3S-[Tyr(SO3Na)]n-ONa (n = 8-19), from reaction mixtures of tyrosine with sulfur trioxide trimethylamine and pyridine comlexes, respectively, in pyridine. Their anti-HIV activity increased along with the increase of the chain length up to the dodecamer, maintained the same level to the length of the heptadecamer and then decreased. The maximal activity level was the same as or higher than that of dextran and curdlan sulfates.

Characterization of Temperate Bacteriophage Bα Isolated from Streptomyces lavendulae

An actinophage designated Bα was isolated from Streptomyces lavendulae S283. Phage Bα produced turbid plaques characteristic of temperate phage on S. lavendulae and other Streptomyces strains. Electron microscopic observation showed that phage Bα belongs to group B of Bradleys morphological classification. Surviving cells from the plaques were lysogenic: they liberated phages during growth and were not lysed by the phage. Phage Bα was immediately inactivated on dilution into NaCl unless Ca2+ was also present. The cleavage pattern of the phage DNA was studied using restriction endonucleases.

A specific cephalosporin-binding protein ofCitrobacter freundii

1. A cephalosporin-binding protein obtained from a strain of Citrobacter freundii was purified to the extent of a single band in analytical and sodium dodecyl sulfate-containing disc electrophoresis. 2. The molecular weight determined by disc electrophoresis was 53 000. 3. The binding protein did not show any beta-lactamase activity at substrate concentrations examined: 6 mM to 100 muM of penicillins and 12 mM to 100 muM of cephalosporins. 4. In gel filtration, [14C]benzylpenicillin was found not to bind to the binding protein. 5. In fluorescence titration, all cephalosporins tested quenched the fluorescence. Association constants of cephalosporins were in the range of 0.8-12-103 M-1, and one binding site was calculated for all cephalosporins tested.

Penicillin-binding proteins of Escherichia coli. Comparison of a strain carrying an R-factor and the parent strain.

Both from Escherichia coli K12 W3630 carrying an R-factor, R+75, and from the parent strain at least six penicillin- and cephalosporin-binding proteins were obtained as soluble forms. The molecular weights of the binding proteins of the strain carrying an R-factor were similar to those of the parent strain and not affected by the presence of an R-factor which specified the production of a beta-lactamase. Gel filtration with [14C]benzylpenicillin suggested the equimolar binding of benzylpenicillin to each binding protein. Three binding proteins of E. coli carrying R+75 and two binding proteins of the parent strain were purified by affinity chromatography followed by gel filtration. In fluorescence titration, various penicillins and cephalosporins were shown to bind to the purified binding proteins and their association constants were in the range of 0.4 to 21-10(3) M-1. The binding proteins of both strains did not react with the antibody against the beta-lactamase specified by R+75.