Rat‐liver lysosomal sialidase

Abstract

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity.The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60000 k 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides, those containing (a2+ 3)sialyl linkages being better substrates than those with (a2+6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin.In contrast to the intralysosomal silaidase, the sialidase partially purified from rat liver cytosol by (NH,),SO, fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60000. It also hydrolyzed gangliosides but not submaxillary mucin.

Effects of OK-432 activation on the sialidase activities of rat peritoneal macrophages.

“…lA also shows that the profile of the sialidase activity toward 4MU-NeuAc is closely similar to that of acid phosphatase, a marker enzyme for the lysosomes, thereby suggesting that the 4MU-NeuAc sialidase here may be identical, or at least closely similar, to the intralysosomal sialidase previously characterized in rat liver (Miyagi and Tsuiki 1984). The finding that the enzyme is inactive toward GM3 (see above) is also consistent with this suggestion since hepatic intralysosomal sialidase has been shown not to attack gangliosides (Miyagi and Tsuiki 1984) In addition to the sialidase activity toward GM3, Fig. lB shows how the activities of two plasma membrane marker enzymes, 5'-nucleotidase and alkaline phosphatase, fluctuate upon the OK-432 activation of peritoneal macrophages.…”

“…Since more than a single type of sialidase were supposed to be present in this fraction Tsuiki 1984, 1986), the assay was made with 4MU-NeuAc and GM3, the preferential substrate for hepatic intralysosomal (Miyagi and Tsuiki 1984) and plasma membrane sialidase (Miyagi and Tsuiki 1986), respectively. Fig.…”

“…In this study, we have produced a rabbit antiserum against the sialidase of rat brain synaptosomal ; this antibody immunoprecipitated the ganglioside sialidase described above and the sialidases of rat liver plasma membrane and erythrocyte ghost ) almost equally thereby supporting the identity of the three sialidases. The existence of multiple types of sialidase in animal cells is now well established (Miyagi and Tsuiki 1984, 1985, 1986, but their physiological and pathological significance still remains obscure. The present studies have shown that the peritoneal macrophages of the rat express asialo-GM1 subsequently to their activation with OK-432 ( Table 1).…”

“…We have previously characterized three types of sialidase (EC 3.2.1.18) in rat liver : they were designated the intralysosomal, cytosolic and plasma membrane sialidases according to their major subcellular location (Miyagi and Tsuiki 1984, 1985, 1986. In attempts to gain further insights into the roles of these sialidases, we have recently studied how the sialidase activities of peritoneal macrophages of the rat are affected when these cells are activated with Chugai,Tokyo).…”

Multiple Forms of Mammalian Sialidase. Altered Expression in Carcinogenesis.

“…Intralysosomal sialidase was partially purified from the particulate fraction of rat liver as described previously (Miyagi and Tsuiki 1984). Cytosolic sialidase was purified homogeneously from the cytosolic fractions of rat liver or skeletal muscle accordng to the method described in the previous work (Miyagi and Tsuiki 1985).…”