1975 Volume 23 Issue 8 Pages 2558-2563
The β-lactamase from clinically isolated cephalosporin resistant Staphylococcus aureus strain No. 41 has been highly purified and characterized. The enzyme was purified by the following procedures : chromatography on DEAE-Sephadex A-50, chromatography on CM-Sephadex C-25, gel filtration on Sephadex G-100 and disc electrophoresis. From ultracentrifugation and gel filtration on Sephadex G-100, sedimentation coefficient was estimated to be 2. 3 and molecular weight to be 22, 000-24, 000.
The isoelectric point was estimated to be 9.5 by electrofocusing on carrier ampholine. The optional pH range of penicillinase activity showed at around 6, while cephalosporinase activity showed at around 5.
The enzyme inducibility was proportionated to the MIC of the substrate, cephalosporin derivative had very high inducibility of the penicillinase and cephalosporinase activity, especially cephalexin had the highest inducibility,
Iodine inhibited the enzyme activity, acted as a competitive inhibitor to the hydrolysis of penicillin-G, not as a competitive one to the hydrolysis of cephaloridine, also this inhibition was not allosteric one.
The ratio of enzyme activity as penicillinase and cephalosporinase expanded through the purification step. But substrate specificity was not proportionated to the MIC to the substrate, and an existance of other mechanism of resistance to the cephalosporins was suggested.
