PURIFICATION AND CHARACTERIZATION OF N-ACETYLMURAMYL-L-ALANINE AMIDASE FROM HUMAN PLASMA USING MONOCLONAL-ANTIBODIES

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 hum an plasma using a three-step column chromatography procedure. Monoclon al antibodies were produced against the purified human enzyme. By coup ling of a high affinity monoclonal antibody to sepharose beads an immu noadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human pla sma in a single step. SDS-PAGE showed one single band of 70 kDa and tw o-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pi between 6.5 and 7.9. Two different metho ds were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after in cubation with amidase and a colorimetric method when high molecular we ight peptidoglycan was used as a substrate for amidase. It is shown th at 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 substr ate for the amidase. Using one of the monoclonal antibodies against th e amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alan ine amidase is present in granulocytes but not in monocytes from unsti mulated peripheral blood of a healthy donor. The presence of N-acetylm uramyl-L-alanine amidase in granulocytes is a novel finding and perhap s important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.