Abstract
A synthetic nonapeptide, Val-Leu-Ile-Arg-Ile-Met-Val-Ser-Arg, corresponding to residues 286-294 of annexin-II tetramer (A-IIt), was shown to completely inhibit the Ca(2+)-dependent bundling of F-actin by this protein. The inhibitory effect of the nonapeptide required preincubation with F-actin and was reversed by the addition of excess A-IIt. Kinetic analysis suggested that the nonapeptide reduced the K(0.5) but not the Vmax of F-actin bundling. In contrast, addition of excess nonapeptide to A-IIt-bundled F-actin did not reverse F-actin bundle formation. Although the nonapeptide produced a dose-dependent inhibition of A-IIt-dependent F-actin bundling, the binding of A-IIt to F-actin was not affected. These results identify a domain of A-IIt that is involved in the bundling activity of the protein and suggest that this domain binds transiently with F-actin, resulting in activation of the bundling activity of A-IIt.
Highlights
A Nonapeptide to the Putative F-actinBinding Site of Annexing11 Tetramer InhibitsIts Calcium-dependentActivation of Actin Filament Bundling*
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 strated that theannexin I1 tetramer (A-IIt) bound F-actin in the presence of calcium with high affinity and in a cooperative manner
The amino acid residues responsiblefor the F-actin bundling activity of this protein have not been identified, we noticed that a region of A-IIt was homologous to stored at 1 mg/ml (4 "C) prior to use in F-actin bindingbundling the putative F-actin binding region of myosin
Summary
A Nonapeptide to the Putative F-actinBinding Site of Annexing Tetramer InhibitsIts Calcium-dependentActivation of Actin Filament Bundling*. Annexin-I binds F-actin half-maximally at 180 p M calcium (Schlaepfer and Haigler, 1987),whereas A-I1 and A-IIt have been shown to bind and bundle actin filaments half-maximally at millimolar calcium (Gerke and Weber, 1984, 1985;Glenney and Glenney, 1985;Glenney et al, 1987). These latter studiesprovided binding and bundling data only qualitatively, i.e. by either SDS-PAGE or electron microscopy. M.). to generate contractile forces and movements with actin it is
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