It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase 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 chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link 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.
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Research Article|
August 15 1993
Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments
P Graceffa;
P Graceffa
*Department of Muscle Research, Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114
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L P Adam;
L P Adam
†Department of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202
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W Lehman
W Lehman
‡Department of Physiology, Boston University School of Medicine, Boston, MA 02118, U.S.A.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1993 The Biochemical Society, London
1993
Biochem J (1993) 294 (1): 63–67.
Citation
P Graceffa, L P Adam, W Lehman; Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments. Biochem J 15 August 1993; 294 (1): 63–67. doi: https://doi.org/10.1042/bj2940063
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