Sialidase in rabbit blood. Characterization of sialidase purified from rabbit erythrocyte membrane.
Sialidase activities of rabbit blood cells and serum were measured. The leucocyte particulate fraction showed the highest specific activity of sialidase towards mixed gangliosides and sialyllactose, and the cytosolic fraction showed for fetuin. Predominant sialidase activity in the blood was detected in erythrocyte particulate fraction when mixed gangliosides were used as substrate. The sialidase for ganglioside was solubilized from the erythrocyte ghosts by using Triton X-100. The solubilized sialidase was purified 1886-fold by sequential chromatographies on DEAE-cellulose, EAH-Sepharose 4B, Octyl-Sepharose CL-4B, Sephadex G-100, concanavalin-A--Sepharose, N-(p-aminophenyl)oxamic acid-agarose and Heparin-Sepharose CL-6B. The optimum pH of purified sialidase was 4.5 for ganglioside mixture, and this enzyme exhibited M(r) = 48,000 by gel filtration. When the purified sialidase was subjected to SDS/PAGE, a major sialidase-active protein band at M(r) = 54,000 and another fainter inactive protein band with M(r) = 115,000 were observed. The purified enzyme was active towards oligosaccharides, gangliosides, fetuin glycopeptide and 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid except for glycoproteins tested. Fe2+, Fe3+ and dithiothreitol significantly inhibited the enzyme activity, while Triton X-100 activated the enzyme. Inside-out vesicles and unsealed ghosts of rabbit erythrocyte showed the sialidase activity for mixed gangliosides but not for resealed ghosts or intact erythrocytes. These results indicate that the active site of this sialidase is oriented mainly on the inside of the erythrocyte membrane and not on the outside. Treatment of rabbit erythrocyte unsealed ghosts with phosphatidylinositol-specific phospholipase C liberated no sialidase activity toward mixed gangliosides from the ghosts.
Chen
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XG
Oriental Medicine Research Center, Kitasato Institute, Tokyo, Japan.
Nagai
T
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Yamada
H
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D000818:Animals; D001792:Blood Platelets / Q000201:enzymology; D002236:Carbohydrate Conformation; D002412:Cations / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005732:Gangliosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology; D008970:Molecular Weight; D009439:Neuraminidase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D017830:Octoxynol / Q000494:pharmacology; D009844:Oligosaccharides / Q000378:metabolism; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D002412:Cations; D003902:Detergents; D005732:Gangliosides; D009844:Oligosaccharides; D012710:Serum Albumin, Bovine; D017830:Octoxynol; D010738:Type C Phospholipases; D009439:Neuraminidase