Figure 3.

VLN1 Binds to F-Actin in a Calcium-Independent Manner.

(A) A high-speed cosedimentation assay was used to determine VLN1 binding to F-actin. A mixture of 3 μM F-actin and 1 μM VLN1, or 3 μM F-actin and 1 μM FIM1, was centrifuged at 200,000g for 1 h. The resulting supernatants and pellets were subjected to SDS-PAGE and Coomassie-stained. The position of actin, VLN1, and FIM1 are marked at the right. The samples include: lane 1, actin alone total; lane 2, actin alone supernatant; lane 3, actin alone pellet; lane 4, actin plus VLN1 total; lane 5, actin plus VLN1 supernatant; lane 6, actin plus VLN1 pellet; lane 7, VLN1 alone total; lane 8, VLN1 alone supernatant; lane 9, VLN1 alone pellet; lane 10, actin plus FIM1 total; lane 11, actin plus FIM1 supernatant; lane 12, actin plus FIM1 pellet; lane 13, FIM1 alone total; lane 14, FIM1 alone supernatant; lane 15, FIM1 alone pellet.

(B) Increasing concentrations of VLN1 (0.3–3.4 μM) were cosedimented with 3 μM F-actin. The concentration of bound VLN1 was plotted against the concentration of free VLN1 and fitted with a hyperbolic function. For this representative experiment, the calculated dissociation constant (K_d) was 0.99 μM.

(C) Identical experiments to that shown in (B) were performed with FIM1. For this representative experiment, the K_d value was 0.67 μM.

(D) Experiments similar to those described for (A) were repeated (n = 3) and extended to include different free [Ca²⁺]. The resulting gels were scanned to determine the amount of VLN1 that was present in the pellet fraction under each condition. The percentage of VLN1 that cosedimented with F-actin at 4.6 nM Ca²⁺ was 65.0; at 100 nM Ca²⁺ was 67.4; at 1 μM Ca²⁺ was 69.9, at 12 μM Ca²⁺ was 66.1; and at 1 mM Ca²⁺ was 64.2.