Purification and characterization of N-acetylmuramoyl-L-alanine amidase from human serum.
Purification to homogeneity of the N-acetylmuramoyl-L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) from human serum has been achieved with a high yield. By molecular sieving chromatography, a molecular weight of 120,000-130,000 has been found for the native enzyme. Polyacrylamide gel electrophoresis under native conditions gave a unique band of Mr = 125,000. The same technique performed under denaturing conditions revealed that the protein is a dimer composed of one subunit of Mr = 57,000 and another of Mr = 70,000. In isoelectrofocalization assays, the amidase behaved as an acidic protein. Ethylenediaminetetraacetate inhibited the enzyme activity; the Mg2+ requirement was confirmed. The simultaneous presence of sulfhydryl groups and disulfide bonds in the protein was evidenced by the inhibitions produced by different thiol-blocking reagents and by several thiol-bearing substances. Direct measurements established the presence of two accessible thiol groups and the occurrence of nine disulfide bonds per protein molecule. Studies of substrate hydrolyzing capacities showed a marked preference for the muramoyl tripeptide derived from the Escherichia coli or Bacillus cereus mureins, the disaccharide tetrapeptide and the bis disaccharide tetra-tetrapeptide from E. coli were also good substrates. Activities on small muropeptides of other composition are also reported. Whole (insoluble) peptidoglycans representing the main bacterial chemotypes were submitted to the enzyme action; although with weak specific activities, the human amidase was nevertheless able to release soluble peptides from some of them. A bacteriolytic capacity on some microorganisms cannot be excluded. Results are discussed and the human enzyme is compared to presently known microbial muramoyl amidases.
Vanderwinkel
E
E
Institut de Recherches du C.E.R.I.A., Brussels, Belgium.
de Vlieghere
M
M
de Pauw
P
P
Cattalini
N
N
Ledoux
V
V
Gigot
D
D
ten Have
J P
JP
D000581:Amidohydrolases / Q000097:blood*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000097:blood* / Q000302:isolation & purification; D009842:Oligopeptides; D013379:Substrate Specificity
D046911:Macromolecular Substances; D009842:Oligopeptides; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase