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Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface

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Abstract

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK m value of 2.56 · 10−4 m and an apparentV max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface.

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Authors and Affiliations

  1. Department of Medical Chemistry and Biochemistry, The Medical School, University of Milan, Via Saldini 50, 20133, Milan, Italy

    Anna Chiarini, Amelia Fiorilli, Lucia Di Francesco, Bruno Venerando & Guido Tettamanti

Authors
  1. Anna Chiarini
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  2. Amelia Fiorilli
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  3. Lucia Di Francesco
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  4. Bruno Venerando
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  5. Guido Tettamanti
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Additional information

Abbreviations: PLC, phospholipase C; PIPLC, phospholipase C acting selectively on phosphatidylinositol; NeuAc,N-acetylneuraminic acid; MU, 4-methylumbelliferone; PBS, Dulbecco's phosphate buffer saline solution. Gangliosides were coded according to Svennerholm [42] and the IUPAC-IUB recommendations [43].

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Chiarini, A., Fiorilli, A., Di Francesco, L. et al. Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface. Glycoconjugate J 10, 64–71 (1993). https://doi.org/10.1007/BF00731189

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