title: Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units.
abstract: The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.
mesh_terms: D003090:Coliphages / Q000201:enzymology*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009439:Neuraminidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides; D011134:Polysaccharides; D013379:Substrate Specificity
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase