CHARACTERIZATION OF THE REGULATORY DOMAIN OF GIZZARD CALPONIN - INTERACTIONS OF THE 145-163-REGION WITH F-ACTIN, CALCIUM-BINDING PROTEINS, AND TROPOMYOSIN

Earlier, we proposed that the interaction of gizzard calponin with F-a ctin, promoting the inhibition of the actomyosin ATPase activity, invo lves the NH2-terminal portion of the calponin segment Ala(145)-Tyr(182 ) (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 coveri ng different stretches of the sequence Thr(133)-Ile(163). Co-sedimenta tion with F-actin, actomyosin ATPase measurements, and zero-length cro ss-linking reactions demonstrated that the 19-residue sequence Ala(145 )-Ile(163) is essential for actin interaction and ATPase inhibition, F urthermore, 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 Ii-re sidue segment Gln(153)-Ile(163), representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high aff inity 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 spec trofluorometry. 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.