A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling.

Purification of Proteins-Annexin-I, annexin-11, and annexin-IIt were purified from frozen bovine lung according to the procedure of Khanna et al. (1987).All steps were carried out at 4 "C.Purified proteins were dialyzed against 20 mM Tris-HC1, pH 7.5, containing 1 mM DTT and concentrated to 2-3 mg/ml by Amicon PM-10 ultrafiltration.Annexin-IIt was stored at -80 "C while A-I and A-I1 were used fresh.Actin was prepared from fresh rabbit skeletal muscle according to Pardee and Spudich (1982).G-actin was further purified by gel filtration on a Sephadex G-100 column (2.6 X 60 cm, Pharmacia LKB Biotechnology Inc.) equilibrated with G-buffer (2 mM Tris-HCI, pH 7.5, 0.5 mM DTT, 0.1 mM CaCI2, 0.33 mM ATP.Na2, 0.01% NaNs).G-actin was lyophilized in the presence of sucrose (2 mg of sucrose per mg of actin) at -80 'C (Tellam and Frieden, 1982).Lyophilized actin was dissolved in and dialyzed against G-buffer for 1-2 days at 4 "C and then centrifuged at 150,000 X g for 1.5 h before use.G-actin was converted to filamentous actin (F-actin) by addition of KC1 and MgC12 to final concentrations of 50 and 1 mM, respectively, and stored at 1 mg/ml (4 "C) prior to use in F-actin bindingbundling assays.Actin Cosedimentation Assays-Actin binding and bundling assays were performed by cosedimentation with F-actin in bundling buffer (50 mM KCl, 1 mM MgC12, 0.33 mM ATP.Na2, 0.5 mM DTT, 0.4 mM CaC12, 0.2 mM EGTA, 2 mM Tris-HC1, pH 7.5) containing F-actin (1.77 PM final), A-IIt, and nonapeptide in a final volume of 0.1 ml.The F-actin was incubated in bundling buffer in the presence or absence of the nonapeptide for 10 min at room temperature, followed by addition of A-IIt.Mixtures were incubated for 10 min at room temperature prior to analysis.Reaction mixtures were initially assayed for light scattering followed by sedimentation (20 "C) at high speed (20 min, 353,000 X g, TLA-100 rotor, Beckman) to assess F-actin binding or low speed (10 min, 15,600 X g, Eppendorf 5414 centrifuge) to assess F-actin bundling.Supernatants were removed, and pellets were dissolved in 40 pl of 2 X SDS sample disruption buffer (0.25 M Tris-HC1, pH 6.8,