Assay for lytic transglycosylases: a family of peptidoglycan lyases.
An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel. Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time. Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis. The optimal conditions for this acid hydrolysis were established to be incubation at 96 degrees C for 1 h in 6 M HCl, in vacuo. The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer. To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12. The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment. On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid. This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa.
Blackburn
N T
NT
Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Clarke
A J
AJ
D000596:Amino Acids / Q000032:analysis; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002852:Chromatography, Ion Exchange; D016695:Glycosyltransferases / Q000378:metabolism*; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D016695:Glycosyltransferases; C021815:murein transglycosylase; D009113:Muramidase