title: Specific acylation of calmodulin. Synthesis and adduct formation with a fluorenyl-based spin label.
abstract: The spin-labeling reagent, N4-(9'-fluorenylmethyloxycarbonyl)-4-amino-1-oxyl-4-succinimidyloxyca rbonyl- 2,2,6,6-tetramethylpiperidine, and the same enriched in 14C at the 4-formyl group, were synthesized as new acylating compounds for protein amino groups that can preserve charge. Porcine testicular calmodulin was modified with this reagent at pH 7.8 in the presence of Ca2+ under conditions that yielded a fairly homogeneous derivative as judged by electrophoretic analysis and tryptic digestion patterns. The tryptic peptides were separated by gel filtration and reverse-phase high-performance liquid chromatography, and the resulting, highly purified 14C-labeled peptides were hydrolyzed and their amino acid compositions determined. The results indicate that at least 87% of the modifications occur at lysyl residues 75 and 148, and the former appears to be the most reactive. This bilabeled calmodulin adduct does not activate a bovine brain cyclic nucleotide phosphodiesterase preparation. The fluorenylmethyloxycarbonyl portion of this inactive calmodulin derivative can, however, be removed by conditions that do not diminish native calmodulin activity in the phosphodiesterase assay. The resulting calmodulin adduct is active in the enzymic assay, although with diminished potency compared to calmodulin. The specificity of the reaction of this acylating reagent with calmodulin may be due to recognition of the tricyclic fluorene ring by the phenothiazine-binding sites since it was found that trifluoperazine inhibited the labeling reaction. Also, calmodulin was far more reactive to this reagent than were several other proteins. This is the first report of a specific, characterized lysine modification on calmodulin, and it is possible that other phenothiazine-binding proteins may also exhibit similar selectivity for acylation. Electron paramagnetic resonance spectra of the calmodulin adducts suggest a high degree of spin immobilization in both the Ca2+-free and Ca2+-saturated states.
mesh_terms: D000215:Acylation; D000596:Amino Acids / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005449:Fluorenes; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D013113:Spin Labels / Q000138:chemical synthesis*; D013552:Swine; D013737:Testis / Q000378:metabolism; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D002147:Calmodulin; D002250:Carbon Radioisotopes; D005449:Fluorenes; D007202:Indicators and Reagents; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D013113:Spin Labels; D014268:Trifluoperazine; D010727:Phosphoric Diester Hydrolases; D002118:Calcium