Differentiation of bacterial autolysins by zymogram analysis.
The use of zymograms in which the bacterial cell wall heteropolymer peptidoglycan is incorporated into the resolving gel of SDS-PAGE has led to the identification of various SDS stable peptidoglycan hydrolases (autolysins). To examine the specificity of autolysins with respect to O-acetylated peptidoglycan, a discontinuous SDS-PAGE system has been developed that operates under neutral conditions. [Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (Bis-Tris) buffers are employed with pH 6.8 and 6.3 for the separating and stacking gels, respectively, while the anode buffer N-2-acetamido-2-hydroxyethanesulfonic acid (Aces)-HCl and the Bis-Tris cathode buffer both had a pH of 6.8. These conditions resulted in a relative trailing ion mobility of 0.349 and 0.137 in the resolving and staking gel, respectively, under room temperature conditions. Peptides and proteins were resolved in the 3-100 kDa range with a 10% acrylamide resolving gel. Comparison of zymograms that incorporated unacetylated or chemically O-acetylated peptidoglycan revealed the specificity of hen egg-white lysozyme for the unacetylated material. A preliminary analysis of the autolysins produced by the urinary tract pathogen Proteus mirabilis indicated that some enzymes were specific for either O-acetylated or non-O-acetylated peptidoglycan while others displayed no clear preference toward either of the two substrates.
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Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Clarke
A J
AJ
D000107:Acetylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis* / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism*; D011511:Proteus / Q000201:enzymology; D013379:Substrate Specificity / Q000502:physiology*
D010457:Peptidoglycan; D009238:N-Acetylmuramoyl-L-alanine Amidase