Abstract
The peptidoglycan transglycosylase of Bacillus megaterium has been purified approximately 500-fold from a crude membrane fraction. This protein is likely to be the one previously called PG-II and was assayed by its ability to reconstitute with a crude phospho-N-acetyl-muramyl-pentapeptide translocase preparation and partially purified N-acetylglucosaminyl transferase to give peptidoglycan synthesis from nucleotide precursors. The protein was identified as the peptidoglycan transglycosylase by its ability to synthesize lysozyme-sensitive peptidoglycan from undecaprenylpyrophosphoryl-disaccharide-pentapeptide. The enzyme is inhibited by vancomycin but not by bacitracin, penicillin G, or tunicamycin. The enzyme has no detectable transpeptidase activity, but it does bind penicillin.
Highlights
Protoplast membranes of B. megaterium were first extracted with 1.5 M LiCl in T M buffer (20 mMMgC12, 0.05 M Tris-HC1, pH 7.4), for 15 min a t 30 “C to recover the transferase [7, 8]
Peptidoglycan remained at the origin, bactoprenyl intermediates had anRFof approxprotein fromcholate-solubilized membranes called PG-I1 was imately 0.8, and disaccharide-peptide monomers resulting from lysoadded [9]
Purification of PG-ZZ-In previous experiments, PG-I1was prepared from membranes of B. megaterium solubilized with cholate and LiCl [9].This protein was assayed by combining it with crude translocase prepared by 0.1 N KOH extractionof membranes [9] and with partially purified transferase obtained by extracting toluene-treatedB. megaterium cells with LiCl [7,8].The combined enzymes in acholate solution were
Summary
Purification of PG-ZZ-The first three steps in the purification of PG-I1 were essentially as described previously [9]. Coli, these activitiescan becarried outby asingle bifunctional Active fractions were pooled and concentrated by precipitation with enzyme which is penicillin-binding protein [1,2,3,4,5,6]. We began with our finding that the two enzymes yielding the transglycosylase substrate, the phospho-MurNAc-pentapeptide translocase and -acetylglucosaminyl transferase [7, 8],were not able to make peptidoglycan but could do so if a GlcNAc. Analysis of Assay Products-All assay mixtures were analyzed by paper chromatography in isobutyric acid, 1N NHlOH [53]solvent as previously described [9]. Analysis of Assay Products-All assay mixtures were analyzed by paper chromatography in isobutyric acid, 1N NHlOH [53]solvent as previously described [9] Under these conditions, peptidoglycan remained at the origin, bactoprenyl intermediates had anRFof approxprotein fromcholate-solubilized membranes called PG-I1 was imately 0.8, and disaccharide-peptide monomers resulting from lysoadded [9].
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