1988 Volume 36 Issue 10 Pages 4008-4018
Sialidase activity was detected in mouse liver, and was localized predominantly in the lysosomal fraction. Weak activity was also contained in the microsomal fraction, but little was detected in the cytosolic fraction. The optimum pH values of these sialidase activities were 4.0-4.5 for 4-methylumbelliferyl-α-D-N-acetylneuraminate (4-M U-NeuAc) and sialyllactose.
The sialidase activity in the lysosomal fraction was very unstable, but was partially stabilized in the presence of phenylmethylsulfonyl fluoride, Ca2+, Mg2+, Mn2+ and Zn2+.
The lysosomal and microsomal sialidase fractions were active preferentially toward ganglioside mixture, in the presence of sodium cholate as a detergent. These enzymes were also active toward all of the sialooligosaccharides and sialoglycoproteins tested. Those substrates possessing an α(2→3)-sialyl linkage were hydrolyzed much faster than those with an α(2→6) or α(2→8) sialyl linkage. These sialidase fractions were much more active toward glycopeptides than glycoproteins. The microsomal sialidase fraction was also active toward submaxillary mucin (bovine), suggesting that it is capable of hydrolyzing O-acetylated sialic acid residues.
When the lysosomal fraction was disrupted hypotonically, the activity toward 4-MU-NeuAc and sialyllactose was mainly recovered in the 15000×g supernatant, and it was fractionated with ammonium sulfate. The fractionated soluble lysosomal sialidase failed to attack gangliosides, andwas only weakly active toward glycoproteins but was capable of hydrolyzing glycopeptides and oligosaccharides. This enzyme was further purified by chromatography on diethylaminoethyl (DEAE)-cellulose, concanavalin A adsorption, affinity chromatography on Sephadex G-200 and high performance liquid chromatography on coupled columns of TSK-GEL G5000PW and G4000PW. When the soluble lysosomal sialidase fraction was subjected to DEAE-cellulose chromatography, most of the β-galactosidase was eluted in the unadsorbed fraction, but the sialidase was eluted in the unadsorbed and the adsorbed fractions.
