title: Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies.
abstract: N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.
authors.affiliation: Department of Immunology, Erasmus University Rotterdam, The Netherlands.
authors.forename: M A
authors.initials: MA
authors.lastname: Hoijer
authors.forename: M J
authors.initials: MJ
authors.lastname: Melief
authors.forename: W
authors.initials: W
authors.lastname: Keck
authors.forename: M P
authors.initials: MP
authors.lastname: Hazenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000097:blood* / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D009238:N-Acetylmuramoyl-L-alanine Amidase