TABLE 2.
Comparison of cellular disaggregation rates to dissociation rates obtained from experiments using rhVCAM-1 and U937 cells after 0.5 min of stirring at three affinity states of VLA-4
| Condition | Mn2+ | Mn2+ + Ca2+ | Mn2+ + 10 mM Ca2+ | |
|---|---|---|---|---|
| Monomeric ligand | koff – rhVCAM-1 (× 10−4 1/s)* | 140 ± 20 | 360 ± 20 | — |
| t1/2 – rhVCAM-1 (s)* | 50 ± 7 | 19 ± 1 | — | |
| koff – rhVCAM-FITC (× 10−4 1/s)† | 340 ± 50 | 650 ± 11 | (12200 ± 320)‡ | |
| t1/2 – rhVCAM-FITC (s)† | 20 ± 3 | 11 ± 2 | (0.57 ± 0.02)‡ | |
| Live B78H1-U937 | kdisagg – aggregates (× 10−4 1/s) | 183 ± 19 | 393 ± 22 | >2497 |
| (N = 3) | t1/2 – aggregates (s) | 38 ± 4 | 18 ± 1 | <2.8 |
| Fixed B78H1-U937 | kdisagg – aggregates (× 10−4 1/s) | 159 ± 42 | 514 ± 186 | >2088 |
| (N = 3) | t1/2 – aggregates (s) | 44 ± 12 | 13 ± 5 | <3.3 |
| Live CHO-U937 | kdisagg – aggregates (× 10−4 1/s) | 153 ± 65 | 378 ± 43 | >2970 |
| (N = 3) | t1/2 – aggregates (s) | 45 ± 2 | 18 ± 2 | <2.3 |
| Cellular average | kdisagg – aggregates (× 10−4 1/s) | 165 ± 43 | 428 ± 9 | >2518 |
| t1/2 – aggregates (s) | 42 ± 11 | 16 ± 4 | <2.3 (live) | |
| Ratio of single dissociation rate to that of multiple bond dissociation rate | Xf (aggregate-rhVCAM-1) | 0.8 ± 0.2 | 0.8 + 0.1 | — |
| Xf (aggregate-rhVCAM-FITC) | 2.5 ± 0.6 | 1.6 + 0.1 | <4.1 | |
| Number of bonds | (Using rhVCAM-1) | 0.9 ± 0.2 | 0.8 ± 0.1 | — |
| (Using rhVCAM-FITC) | 2.6 ± 1.8 | 1.8 ± 0.1 | <11.6 |
Data obtained from percentage of aggregation with respect to U937 cells and percentage of U937 cells with respect to number of aggregates. The errors reflect the standard error in mean of curves fitted to the data. Number of experimental measurements obtained is denoted by N.
Data from competitive binding experiments using LDV-FITC-containing small molecule as a ligand and nonlabeled rhVCAM-1 as a competitor. Those results represent the natural ligand dissociation rates for monomeric VCAM-1.
Data obtained from rhVCAM-FITC dissociation experiments.
Data obtained using rapid mix flow cytometry (Graves et al., 2002). Monomeric ligand results are data from Chigaev et al. (2003a). Bond number and Xf are upper limits due to the minimum time required to handle samples.