Isolation and characterization of an oligosaccharide- and glycoprotein-specific sialidase from human leucocytes.
A sialidase was solubilized with the aid of Triton X-100 from the insoluble material of a leucocyte homogenate. The enzyme was purified almost to homogeneity by chromatography on Sephadex G-75, equine submandibular gland mucin bound to Sepharose 4B and on Sephacryl S-200. The purification factor was 40 based on an increase of the specific enzyme activity from the Triton X-100 extract (pure enzyme: 40 mU/mg protein). Isolation of the active enzyme required the presence of a proteinase inhibitor. The sialidase is monomeric and has an average molecular mass of 48500 Da, a pH optimum of 4.6, hydrolyses preferably glycoprotein (fetuin) and sialyllactose, is activated by Ca2 and inhibited by N-acetyl-2,3-dehydro-2- deoxyneuraminic acid ( Neu5Ac2en ), Hg2 and N-(4-nitrophenyl) oxamic acid. The relatively stable enzyme shows only low activity with gangliosides and no activity with 4-O-acetylated sialic acid bound glycosidically.
D001798:Blood Proteins / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D004589:Electrophoresis, Disc; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007962:Leukocytes / Q000201:enzymology*; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives; D009438:Neuraminic Acids / Q000378:metabolism; D009439:Neuraminidase / Q000097:blood / Q000302:isolation & purification*; D009844:Oligosaccharides / Q000097:blood; D012794:Sialic Acids / Q000378:metabolism; D013379:Substrate Specificity
D001798:Blood Proteins; D006023:Glycoproteins; D009438:Neuraminic Acids; D009844:Oligosaccharides; D012794:Sialic Acids; C032592:N-glycolylneuraminic acid; C041901:4-O-acetyl-N-acetylneuraminic acid; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid