DISULFIDE CROSS-LINKING OF SMOOTH-MUSCLE AND NONMUSCLE CALDESMON TO THE C-TERMINUS OF ACTIN IN RECONSTITUTED AND NATIVE THIN-FILAMENTS

It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 c an be disulphide-cross-linked to the C-terminal penultimate residue (C ys-374) of actin, indicating that these residues are close in the prot ein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20 305-20310]. Since the possibility that the cross-link involves a cyste ine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identity of the cysteine residues involved in the cross-link. We show here that caldesmon could not be d isulphide-cross-linked to actin which had Cys-374 removed by carboxype ptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chick en gizzard caldesmon, which has an additional cysteine residue at posi tion 153. The porcine stomach caldesmon also formed a disulphide-cross -link to actin, further supporting the original conclusion that Cys-58 0 of the chicken gizzard caldesmon had been cross-linked to actin. Dis ulphide-cross-linking with similar yield was also observed in native c hicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in na tive and reconstituted thin filaments. The much smaller non-muscle iso form of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-termin al domain of muscle and non-muscle caldesmon. The ability to cross-lin k caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.