Drug protein interactions: isolation and characterization of covalent adducts of phenoxybenzamine and calmodulin

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Abstract
Phenoxybenzamine, an .alpha.-adrenergic antagonist containing a (chloroethyl)amine group, labels calmodulin the presence of Ca. The covalent interaction is inhibited by chloropramazine in a concentration-dependent manner. Adducts of calmodulin and phenoxybenzamine were separated by high-performance liquid chromatography into the following 4 major fractions: 2 containing 9.6 and 1.2 mol of drug/mol of protein and 2 different fractions each containing 2.0 mol/mol. Each adduct had a reduced ability to active cyclic nucleotide phosphodiesterase and myosin light chain kinase. The chlorpromazine binding capcities of the phenoxybenzamine calmodulin adducts were diminished to the extent of phenoxybenzamine incorporation into each adduct. Isolation and characterization of labeled peptides from phenoxybenzamine-modified calmodulins indicated that peptides encompassing residues 38-75, 107-126, and 127-148 contained phenoxybenzamine label. These studies directly demonstrate the relatedness between the binding activities of 2 structurally dissimilar calmodulin antagonists, demonstrate that covalent adducts of calmodulin and drugs with equal stoichiometries of labeling can have quantitative differences in activity and sites of modification and provide direct evidence of distinct drug binding regions in calmodulin located in the amphipathic .alpha.-helical regions of the 2nd and 4th domains.
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