Topographical mapping of calmodulin-target enzyme interaction domains.
Calmodulin derivatives, specifically biotinylated in domains I and III, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'-cyclic nucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-Mg2+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+2 binding region of domain III. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.
Mann
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Graduate Center for Toxicology, Albert B. Chandler Medical Center, University of Kentucky, Lexington 40536.
Vanaman
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D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D008297:Male; D010449:Peptide Mapping; D011485:Protein Binding; D013737:Testis / Q000378:metabolism; D014908:Triticum / Q000378:metabolism; D014357:Trypsin
D002147:Calmodulin; D001360:Avidin; D001710:Biotin; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases