Preparation and characterization of biotinylated analogs of the calcium channel blocker omega-conotoxin.
We have prepared a series of biotinylated analogs of omega-conotoxin (omega CgTx) as potent, selective markers for N-type calcium channels. At pH 9.5, reaction of omega CgTx with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24, or at both positions. Kinetic competition assays of 125I-omega CgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 x 10(6) M-1 s-1) but not dissociation (1-4 x 10(-4) s-1). Comparison with rate constants obtained for the association (1.2 x 10(7) M-1 s-1) and dissociation (5 x 10(-5) s-1) of native omega CgTx indicates that while biotinylation reduces omega CgTx potency (Kdkin = k-2/k2 = 4 pM for omega CgTx), binding of these labels to membranes is nevertheless of very high affinity (Kdkin 0.1-0.3 nM).
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Playfair Neuroscience Unit, Toronto Western Hospital, Ontario, Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D001921:Brain / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000138:chemical synthesis* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007457:Iodine Radioisotopes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism*; D013816:Thermodynamics; D014357:Trypsin; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D010455:Peptides; D010456:Peptides, Cyclic; D020960:omega-Conotoxins; C053423:Conus magus toxin; D001710:Biotin; D014357:Trypsin