Direct Assay of Sialidase on Intact Human Erythrocytes
| Substrate . | pH . | Sialidase Activity . | |||
|---|---|---|---|---|---|
| . | . | nmol Released Sialic Acid/h/μmol P . | μU/μmol P . | nmol Released Sialic Acid/1 × 107 Cells . | μU/1 × 107 Cells . |
| GD1a | 4.7 | 0.74 ± 0.05 | 12.3 ± 0.83 | 1.23 ± 0.09 | 20.5 ± 1.5 |
| GM3 | 4.7 | 1.85 ± 0.2 | 30.8 ± 3.3 | 3.0 ± 0.21 | 50.0 ± 3.3 |
| GD1a | 7.2 | 0.14 ± 0.02 | 2.3 ± 0.33 | 0.17 ± 0.02 | 2.8 ± 0.33 |
| GM3 | 7.2 | 0.39 ± 0.04 | 6.5 ± 0.66 | 0.64 ± 0.08 | 10.6 ± 1.3 |
| Substrate . | pH . | Sialidase Activity . | |||
|---|---|---|---|---|---|
| . | . | nmol Released Sialic Acid/h/μmol P . | μU/μmol P . | nmol Released Sialic Acid/1 × 107 Cells . | μU/1 × 107 Cells . |
| GD1a | 4.7 | 0.74 ± 0.05 | 12.3 ± 0.83 | 1.23 ± 0.09 | 20.5 ± 1.5 |
| GM3 | 4.7 | 1.85 ± 0.2 | 30.8 ± 3.3 | 3.0 ± 0.21 | 50.0 ± 3.3 |
| GD1a | 7.2 | 0.14 ± 0.02 | 2.3 ± 0.33 | 0.17 ± 0.02 | 2.8 ± 0.33 |
| GM3 | 7.2 | 0.39 ± 0.04 | 6.5 ± 0.66 | 0.64 ± 0.08 | 10.6 ± 1.3 |
Assays were performed at pH 4.7 and 7.2 using 25 to 40 μL of an erythrocyte suspension carrying 8.08 × 106 cells/mL. The radiochemical assay method was used with [3H]GD1a and [3H]GM3 as substrates. At the end of incubation, an aliquot of the mixture was withdrawn and centrifuged, and the hemoglobin content was determined in the supernatant. The degree of hemolysis was in the range of 1.83% to 1.96%. For details, see Materials and Methods. The data are the mean values of five experiments ± SD values.