Purification and characterization of an 85 kDa talin‐binding fragment of vinculin

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin‐binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125 I‐talin and the 125 I‐85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin‐binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125 I‐talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment, was about three fold more effective at competing for 125 I‐85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin‐talin interaction even though the talin‐binding domain is localized in the 85 kDa fragment.