Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain.
A membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100,000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4,851.3 micromol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.
Hata
K
K
The Division of Biochemistry, Research Institute, Miyagi Prefectural Cancer Center, Natori, Miyagi 981-1293, Japan.
Wada
T
T
Hasegawa
A
A
Kiso
M
M
Miyagi
T
T
D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D019826:Cholic Acid; D002793:Cholic Acids; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005732:Gangliosides / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008566:Membranes; D008970:Molecular Weight; D009439:Neuraminidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013574:Synaptosomes / Q000201:enzymology
D002793:Cholic Acids; D005732:Gangliosides; D006023:Glycoproteins; D009844:Oligosaccharides; D017830:Octoxynol; C036052:GM3 ganglioside sialidase; D009439:Neuraminidase; D019826:Cholic Acid