Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone.

A partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced. Expression of the clone in Escherichia coli produced a peptide called H1 (M(r) 32,549), which inhibited tropomyosin-enhanced actomyosin Mg(2+)-ATPase activity by 90% with half maximal inhibition at 0.03-0.04 mol H1 per mol actin. The inhibition could be reversed by Ca(2+)-calmodulin. H1 bound actin, Ca(2+)-calmodulin and tropomyosin and smooth muscle myosin with high affinities. This latter finding shows the presence of a second myosin-binding site in caldesmon. This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.