title: Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.
abstract: A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.
authors.affiliation: Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
authors.forename: R
authors.initials: R
authors.lastname: Kariyama
authors.forename: G D
authors.initials: GD
authors.lastname: Shockman
mesh_terms: D000911:Antibodies, Monoclonal; D016983:Enterococcus / Q000201:enzymology*; D006146:Guanidines; D007527:Isoenzymes; D009113:Muramidase / Q000276:immunology / Q000378:metabolism*; D011489:Protein Denaturation; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D000911:Antibodies, Monoclonal; D006146:Guanidines; D007527:Isoenzymes; D011509:Proteoglycans; D009113:Muramidase