Lukas T J, Marshak D R, Watterson D M
Biochemistry. 1985 Jan 1;24(1):151-7. doi: 10.1021/bi00322a021.
Phenoxybenzamine, an alpha-adrenergic antagonist containing a (chloroethyl)amine group, labels calmodulin in the presence of calcium. The covalent interaction is inhibited by chlorpromazine in a concentration-dependent manner. Adducts of calmodulin and phenoxybenzamine were separated by high-performance liquid chromatography into four major fractions: two containing 0.6 and 1.2 mol of drug per mol of protein and two different fractions each containing 2.0 mol/mol. Each adduct had a reduced ability to activate cyclic nucleotide phosphodiesterase and myosin light chain kinase, and the chlorpromazine binding capacities 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 two 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 second and fourth domains.
酚苄明是一种含有（氯乙基）胺基团的α-肾上腺素能拮抗剂，在有钙存在的情况下可标记钙调蛋白。氯丙嗪以浓度依赖的方式抑制这种共价相互作用。钙调蛋白与酚苄明的加合物通过高效液相色谱法分离为四个主要部分：两个部分每摩尔蛋白质含有0.6摩尔和1.2摩尔药物，另外两个不同部分每摩尔分别含有2.0摩尔药物。每种加合物激活环核苷酸磷酸二酯酶和肌球蛋白轻链激酶的能力均降低，并且酚苄明-钙调蛋白加合物的氯丙嗪结合能力随着酚苄明掺入每种加合物的程度而降低。从酚苄明修饰的钙调蛋白中分离和鉴定标记的肽表明，包含第38 - 75位、第107 - 126位和第127 - 148位残基的肽含有酚苄明标记。这些研究直接证明了两种结构不同的钙调蛋白拮抗剂结合活性之间的相关性，表明具有相同化学计量标记的钙调蛋白与药物的共价加合物在活性和修饰位点上可能存在定量差异，并提供了位于第二和第四结构域两亲性α-螺旋区域的钙调蛋白中不同药物结合区域的直接证据。
