title: Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum.
abstract: Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM.TnI.TnT ternary complex. This suggests that both subunits are within 10 A of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with Kd = 6 x 10(-8) M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.
mesh_terms: D000818:Animals; D001577:Benzophenones / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine; D004910:Erythrocyte Membrane / Q000201:enzymology; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008301:Maleimides / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D014908:Triticum / Q000378:metabolism; D014336:Troponin / Q000378:metabolism*
chemical_list: C032162:AEDANS-calmodulin; D001577:Benzophenones; D002147:Calmodulin; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008301:Maleimides; D009282:Naphthalenesulfonates; D014336:Troponin; C046109:benzophenone-4-maleimide; D000252:Calcium-Transporting ATPases; D003545:Cysteine