TABLE II.
Assembly of vitronectin and PAI-1 into intermediate and higher order complexes. Ranges in s values were defined for integration according to the criteria outlined in Table I, with species sedimenting with s values in the range from 1 to 5 S representing free vitronectin and PAI-1, species with s values in the range from 5.1 to 7.4 S representing intermediate 1:1 and 2:1 complexes, and species sedimenting with s values greater than 7.5 S representing higher order complexes of vitronectin and PAI-1.
| Protein samples | Protein concentrations | Unbound proteins (fraction in s range from 1to5 S)a | Intermediate complexes (fraction in s range from 5.1 to 7.4 S)a | Higher order complexes (fraction in s range from 7.5 to 25 S)a |
|---|---|---|---|---|
| % | % | % | ||
| Vitronectin + wild-type PAI-1 | 8 μM vitronectin 8 μM PAI-1b | 26 | 24 | 50 |
| Vitronectin + wild-type PAI-1 | 3.2 μM vitronectin 3.2 μM PAI-1b | 25 | 45 | 30 |
| Vitronectin + wild-type PAI-1 | 1.6 μM vitronectin 1.6 μM PAI-1b | 30 | 64 | 6 |
| Vitronectin + Y175F PAI-1 | 3.3 μM vitronectin 3.3 μM PAI-1 | 25 | 34 | 41 |
| Vitronectin + PAI-1R | 3.3 μM vitronectin 3.3 μM PAI-1 | 20 | 40 | 40 |
Sedimentation data were evaluated as c(s) profiles, and data were integrated using SEDFIT to determine the relative amounts of various PAI-1-vitronectin complexes formed in the three mixtures. Values were normalized by dividing the integrated values by the total loading signal in fringes and multiplying by 100 to obtain a percentage of the various species formed. Errors are ± 5%.
c(s) distributions of complexes with s values >5 S for the three concentrations of vitronectin and wild-type PAI-1 samples are shown in Fig. 3, B—D. Free proteins that sediment in the 1—5 S range are not shown in the figure, but the integrations over this s range are shown here.