Partial purification and characterization of N-acetylmuramyl-L-alanine amidase from human and mouse serum.

The enzyme N-acetylmuramyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) has been detected in human, mouse, rabbit, bovine and sheep sera. A method for detection of amidase activity using [14C]peptidoglycan monomer as the substrate has been developed. Partial purification of human and mouse amidase was achieved by gel chromatography on Bio-Gel A-1.5 m, DEAE-Sephadex A-50 and Sephadex G-100. Both amidase preparations exhibited maximal activity at pH 9.0 in Tris-HCl buffer and required Mg2+ for maximal activity. Following digestion of peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala, the disaccharide GlcNAc-MurNAc and the corresponding pentapeptide L-Ala-D-isoglutaminyl-meso-diaminopimelyl-D-Ala-D-Ala were formed and subsequently isolated and chemically characterized. The enzyme therefore acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine. The glycan linked, peptide-not-cross-linked peptidoglycan dimer was also shown to be a substrate for human and mouse amidase.