[16] Penicillinase secretion in Vivo and in Vitro

The study of penicillinase secretion has contributed extensively to knowledge of secretory processes in bacteria. In gram-negative bacteria the R6K penicillinase is found in the periplasm. The gram-positive penicillinases occur in two forms—hydrophilic exoenzyme and hydrophobic membrane-bound enzyme...

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Veröffentlicht in:Methods in Enzymology 1983, Vol.97, p.153-158
1. Verfasser: Nielsen, Jennifer B.K.
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Sprache:eng
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Zusammenfassung:The study of penicillinase secretion has contributed extensively to knowledge of secretory processes in bacteria. In gram-negative bacteria the R6K penicillinase is found in the periplasm. The gram-positive penicillinases occur in two forms—hydrophilic exoenzyme and hydrophobic membrane-bound enzyme—encoded by the same gene. The three are identified as lipoproteins of the thioether type. The chapter focuses on the synthesis and isotopic labeling in vivo of the lipoprotein membrane-bound forms of gram-positive penicillinases, and the synthesis both in permeablized cells and in cell-free extracts of full-length translation products. The labeling procedures, though developed for penicillinases in gram-positive organisms, are applicable to the study of thioether lipoproteins in a wide range of bacteria. For three organisms—Bacillus licheniformis, Bacillus cereus, and Staphylococcus aureus—penicillinase production is induced by adding the pseudo gratuitous inducer 2-(2′-carboxyphenyl)benzoyl-6-aminopenicillanic acid to a culture in early exponential phase. Labeling is terminated by adding an excess of the appropriate unlabeled compound.
ISSN:0076-6879
1557-7988
DOI:10.1016/0076-6879(83)97129-X