Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay.
AUTOR(ES)
Roychoudhury, S
RESUMO
We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=163476Documentos Relacionados
- Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.
- Altered penicillin-binding proteins in methicillin-resistant strains of Staphylococcus aureus.
- In vitro effects of beta-lactams combined with beta-lactamase inhibitors against methicillin-resistant Staphylococcus aureus.
- Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins.
- Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography.
