Biochemical and immunological studies on two distinct ganglioside-hydrolyzing sialidases from the particulate fraction of rat brain.
Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.
Miyagi
T
T
Biochemistry Laboratory, Tohoku University, Miyagi.
Sagawa
J
J
Konno
K
K
Handa
S
S
Tsuiki
S
S
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D006868:Hydrolysis; D008297:Male; D009439:Neuraminidase / Q000276:immunology / Q000378:metabolism*; D009928:Organ Specificity; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology*; D013379:Substrate Specificity
D000906:Antibodies; C036052:GM3 ganglioside sialidase; D009439:Neuraminidase