Affinity labeling of calmodulin-binding components in canine cardiac sarcoplasmic reticulum.
Canine cardiac sarcoplasmic reticulum vesicles have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated a 125I-containing major product of Mr = 40,000, and a minor component of Mr = 120,000. This latter component probably represents a 1:1 complex between the 100,000-dalton Ca2+-ATPase protein and 125I-calmodulin. When cross-linked samples, solubilized in sodium dodecyl sulfate, were boiled for 2 min, the radioactivity associated with the 40,000-dalton component decreased while that associated with components of 26,000 and 28,000 daltons increased. When these boiled samples were stored at -70 degrees C for 1 week, the radioactivity associated with the 26,000- and 28,000-dalton components decreased whereas that associated with the 40,000-dalton component was increased. This suggests that the 40,000-dalton component represents a 1:1 cross-link between the 23,000-dalton form of phospholamban and 125I-calmodulin. The 26,000- and 28,000-dalton cross-linked components probably represent 1:1 cross-links between 125I-calmodulin and the 8,000- and 11,000-dalton subunits, respectively, of phospholamban. A 32P-containing, 40,000-dalton component was formed when dithiobis(succinimidyl propionate) was added to sarcoplasmic reticulum vesicles, phosphorylated in the presence of [gamma-32P]ATP and 3 microM calmodulin. This confirms that the 40,000-dalton affinity-labeled component is a 1:1 cross-link between phospholamban and calmodulin. We propose that phospholamban is the endogenous receptor for calmodulin in cardiac sarcoplasmic reticulum membranes. However, it is unlikely that phospholamban is the endogenous calmodulin-dependent protein kinase in this membrane.
D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003432:Cross-Linking Reagents; D004285:Dogs; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013388:Succinimides
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D013388:Succinimides; C011240:dithiobis(succinimidylpropionate)