Isolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive D-alanyl-D-alanine-cleaving transpeptidase.
The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1.
D001426:Bacterial Proteins; D002268:Carboxypeptidases / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010400:Penicillin G / Q000494:pharmacology*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002593:Cetrimonium Compounds; D003902:Detergents; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G; D000077286:Cetrimonium