HUMAN ERYTHROCYTE SIALIDASE IS LINKED TO THE PLASMA-MEMBRANE BY A GLYCOSYLPHOSPHATIDYLINOSITOL ANCHOR AND PARTLY LOCATED ON THE OUTER SURFACE

Treatment of human erythrocyte ghosts with phosphatidylinositol-phosph olipase C (PIPLC) from Bacillus cereus liberated the ghost-linked sial idase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37-degrees-C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the b eginning of incubation and every 15 min (four subsequent additions). L iberated sialidase was fully resistant to at least four cycles of rapi d freezing and thawing and to storage at 4-degrees-C for at least 48 h . The liberated enzyme had an optimal activity at pH 4.2, degraded gan glioside GD1a better than methylumbelliferyl N-acetylneuraminic acid ( about fourfold), and gave a K(m) value of 2.56 x 10(-4) M and an appar ent V(max) of 2.22 mU per mg protein on GD1a. Treatment of intact eryt hrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused l iberation of 10-12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the eryth rocyte membrane. It is concluded that the erythrocyte membrane sialida se is anchored by a glycosylphosphatidylinositol structure sensitive t o PIPLC action, and is partly located on the outer surface.