Binding of caldesmon to smooth muscle myosin.
Caldesmon, a major calmodulin binding protein, was found to bind smooth muscle myosin. Addition of caldesmon to smooth muscle myosin induced the formation of small aggregates of myosin in the absence of Ca2+-calmodulin, but not in the presence of Ca2+-calmodulin. The binding site of myosin was studied by using caldesmon-Sepharose 4B affinity chromatography. Subfragment 1 was not retained by the column, while heavy meromyosin and subfragment 2 were bound to the caldesmon affinity column in the absence of Ca2+-calmodulin but not in its presence. It was therefore concluded that the binding site of caldesmon on myosin molecule was the subfragment 2 region and that binding of caldesmon to myosin was abolished in the presence of Ca2+ and calmodulin. Cross-linking of actin and myosin mediated by caldesmon was studied. While actomyosin was completely dissociated in the presence of Mg2+-ATP, the addition of caldesmon caused aggregation of the actomyosin. By low speed centrifugation at which actomyosin alone was not precipitated in the presence of Mg2+-ATP, the aggregate induced by caldesmon was precipitated and the composition of the precipitate was found to be actin, caldesmon, and myosin. In the presence of Mg2+-ATP, pure actin did not bind to a myosin-Sepharose 4B affinity column, while all of the actin was retained when the actin/caldesmon mixture was applied to the column. These results indicate that caldesmon can cross-link actin and myosin.
Ikebe
M
M
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Reardon
S
S
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000032:analysis*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D013053:Spectrophotometry; D014422:Turkeys
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D009218:Myosins; D002118:Calcium