Eur. J. Biochem. zyxwvutsrqp 157, zyxwvutsrqpon 101 -106 (1986) zyxwvuts 8 FEBS 1986 zyxwvutsrqpo Antibodies against the benzylpenicilloyl moiety as a probe for penicillin-binding proteins Regine HAKENBECK, Thomas BRIESE and Heinz ELLERBROK Abteilung Trautner, Max-Planck-Institut fur molekulare Genetik, Berlin (Received January 13, 1986) - EJB 860032 Antibodies against the benzylpenicilloyl determinant were used to identify complexes of benzylpenicilloyl and penicillin binding protein (PBP) of several bacterial species on immunoblots. Since radioactive penicillin was not needed, this technique readily allowed zyxwvuts in vivo labeling studies even in Escherichia coli, where the saturating concentration was around 0.6 mg/ml. The antibodies showed no substantial cross-reactivity to other /3-lactam - PBP complexes with the exception of 6-aminopenicillanic acid. Surprisingly, some penicilloyl-PBP were hardly recognized by the antiserum, wherease the others could be stained according to the amount of penicillin bound. All penicillin-sensitive bacteria contain membrane-bound penicillin binding proteins, which are able to interact with fl-lactams by forming a covalent antibiotic-enzyme complex. The p-lactam is thought to block the catalytic site of these enzymes, which normally play a role in murein metabolism (for review, see [l, 21). Binding occurs via acylation of a serine residue, as has been shown in numerous cases [3 - 61, even for the somehow related p-lactamases [A. Upon binding to PBP, the p-lactam ring is enzymatically hydrolyzed resulting in a proposed penicilloyl-PBP complex. Supporting evidence comes from investigations, where penicilloic acid has been identified after enzymatic release from PBP of Proteus mirabilis zyxwvutsrqp [8] and Streptococcus faecalis [9]. Penicilloyl- hydroxamate was found after reversal of penicillin binding in Bacillus subtilis membranes with hydroxylamine [lo]. Alkaline hydrolysis of a j-lactam-containing peptide of the high-M, PBP from Escherichia coli has yielded penicilloic acid at least as one compound [ll]. One antigenic determinant that gives rise to the antibody- dependent penicillin-allergic reaction in humans has been shown to be the penicilloyl group of /3-lactam antibiotics [I2 - 151. Antibodies against the penicilloyl moiety can easily be raised in animals, using p-lactam - protein conjugates as anti- gens [13, 161. Thus, the benzylpenicilloyl-PBPcomplex should be recognized by the anti-BPO antibodies. Until now, PBP have been identified after incubation with a radioactive /3-lactam, subsequent SDS-PAGE and fluoro- graphy [17]. In this paper, we will describe a technique by which PBP can be visualized on immunoblots after labeling with nonradioactive benzylpenicillin zyxwvut in vivo or in vitro, using Correspondence to R. Hakenbeck, Abteilung Trautner, Max- Planck-Institut fur Molekulare Genetik, IhnestraDe 63 - 73, D-1000 Berlin 33, Federal Republic of Germany Abbreviations. PBP, penicillin-binding protein; BPO, benzyl- penicilloyl; bIgG, bovine immunoglobulin G; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, physio- logically buffered saline; (LYS)~,,,poly(L-lysine) with 20 residues per chain. anti-BPO antibodies. In this system, differences concerning the structure of the PBP-bound penicillin residue became apparent. MATERIALS AND METHODS Bacterial strains and growth conditions The following strains and media were used: Escherichia coli K12 strain C600 (lacY1, lambda-, leug6, supE44, thi-1, thr-I, tonA21); Bacillus subtilis SB 1207 (r-m- su3+ leu- metB5- thr-) [18]; both strains were grown aerobically in tryptone yeast at 37°C. Streptococcus pneumoniae R6 is a derivative of the Rockefeller University laboratory strain R36A. Cells were grown in C medium [19], supplemented with 0.1% yeast extract at 37°C without shaking. Preparation zyxwvu of membranes Cells of exponentially growing cultures were harvested by centrifugation and washed once with 20 mM sodium phosphate buffer, pH 7.2. Cells were broken in a French pres- sure cell at 137.8 MPa, or in the case of in vivo labeled E. coli, by sonication (three 30-s periods). Membranes were collected at 60000 rpm for 3 h in a Beckman Ti75 rotor. After washing with phosphate buffer, membranes were stored at - 20°C at a concentration of 20 - 60 mg/ml as determined by the method of Lowry et al. [20]. Membranes of Staphylococcus aureus H were kindly provided by F. Beise. Labeling of PBP with antibiotics Membranes were labeled with different antibiotics as de- scribed in the figure legends. Ben~yl[~~S]penicillin (2 - 5 Ci/ mmol) and N-succinimidyl [2,3-3H]propionate (60 - 90 Ci/ mmol) used for the synthesis of [3H]-propionyl ampicillin [21] were from New England Nuclear; [ ''C]benzylpenicillin (50 Ci/mol) and ['H]benzylpenicillin (20 Ci/mmol) were from Amersham. For E. coli PBP, proteins were solubilized at room