Résumé
Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-termi-nal portion of the calponin segment Ala145-Tyr182(Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca2+-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.