Disulfide cross-linking of caldesmon to actin.
Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.
Graceffa
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Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Jancsó
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D000199:Actins / Q000378:metabolism*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000737:chemistry; D009130:Muscle, Smooth / Q000737:chemistry; D009218:Myosins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D000199:Actins; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D005456:Fluorescent Dyes; D004228:Dithionitrobenzoic Acid; D013917:Thrombin; D009218:Myosins