J. Biochem, 1990, Vol. 107, No. 5 787-793
© 1990 Japanese Biochemical Society
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Biochemical and Immunological Studies on Two Distinct Ganglioside-Hydrolyzing Sialidases from the Particulate Fraction of Rat Brain1
*Biochemistry Laboratory, Research Institute for Tuberculosis and Cancer, Tohoku University Aoba-ku, Sendari, Miyagi 980
**Department of Biochemistry, Tokyo Medical and Dental University Bunkyo-ku, Tokyo 113
To whom correspondence should be addressed.
Ganglioside-hydrolyzing sialidases 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 designatede sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on OctyI-Sepharose CL-4B, PhenyI-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 Mi = 70, 000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyI-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.
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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