Preparation, characterization and biological properties of biotinylated derivatives of calmodulin.
Biotinylated derivatives of calmodulin (CaM) were prepared and their biological properties characterized by using enzyme assays, affinity and hydrophobic-interaction chromatography. Several N-hydroxysuccinimidobiotin derivatives [sulphosuccinimidobiotin (sulpho-NHS) and sulphosuccinimido-6-(biotinamido)hexanoate (BNHS-LC)] differing in spacer arm length were used to modify CaM. The shorter-spacer-arm CaM derivative (sulpho-CaM) activated CaM-dependent cyclic nucleotide phosphodiesterase and CaM-dependent protein kinase II; preincubation with avidin blocked its ability to activate these enzymes. The extended-spacer-arm derivative (BNHS-LC-CaM) activated CaM-dependent enzymes both in the presence and in the absence of avidin, suggesting that the longer spacer arm diminished steric effects from avidin preincubation. Other biotinylated CaM derivatives were prepared with biotinylated tyrosine and/or histidine residues (diazobenzoylbiocytin; DBB-CaM) or nucleophilic sites (photobiotin acetate; photo-CaM). These derivatives activated CaM-dependent enzymes in the presence and in the absence of avidin. Oriented affinity columns were constructed with covalently immobilized avidin complexed to each biotinylated CaM derivative. The chromatographic profiles obtained revealed that each column interacted with a specific subset of CaM-binding proteins. Elution profiles of biotinyl CaM derivatives on phenyl-Sepharose hydrophobic-interaction chromatography suggested that several derivatives displayed diminished binding to the matrix in the presence of Ca2+. Development and characterization of a series of biotinylated CaM molecules can be used to identify domains of CaM that interact with specific CaM-dependent enzymes.
Polli
J W
JW
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033.
Billingsley
M L
ML
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D001710:Biotin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D015394:Molecular Structure; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats
D002147:Calmodulin; D001710:Biotin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases