Vorherr T, Knöpfel L, Hofmann F, Mollner S, Pfeuffer T, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
Biochemistry. 1993 Jun 15;32(23):6081-8. doi: 10.1021/bi00074a020.
Peptides corresponding to regions of the calmodulin-activated NO-synthase and of the calmodulin dependent adenylyl cyclase, which could represent the calmodulin binding domains of the two proteins, have been synthesized and tested for calmodulin binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the calmodulin binding domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity calmodulin binding domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylcalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity calmodulin binding site of the calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylcalmodulin and by the ability to inhibit the calmodulin-stimulated activity of purified calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane domain of the molecule. It has the typical hydrophobic/basic composition of canonical calmodulin binding domains, and also contains, like most calmodulin binding domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the calmodulin binding domain of this enzyme.
已合成了与钙调蛋白激活的一氧化氮合酶及钙调蛋白依赖性腺苷酸环化酶区域相对应的肽段，这些肽段可能代表这两种蛋白质的钙调蛋白结合结构域，并对其进行了钙调蛋白结合测试。所选肽段是这两种蛋白质序列中与公认的钙调蛋白结合结构域一般特性最相符的，即含有碱性氨基酸的疏水序列。对于一氧化氮合酶，通过尿素凝胶电泳和以丹磺酰钙调蛋白作为肽段NO - 30（氨基酸725 - 754）的荧光光谱法，鉴定出了假定的高亲和力钙调蛋白结合结构域。通过用丹磺酰钙调蛋白滴定以及抑制纯化的钙调蛋白刺激的腺苷酸环化酶的钙调蛋白刺激活性的能力，将钙调蛋白依赖性腺苷酸环化酶的最高亲和力钙调蛋白结合位点指定给肽段AC - 28（氨基酸495 - 522）。序列495 - 522位于分子第六个假定跨膜结构域向胞质溶胶突出的单元中。它具有典型的钙调蛋白结合结构域的疏水/碱性组成，并且像大多数钙调蛋白结合结构域一样，在其N端部分含有一个芳香族氨基酸。它在中部还含有两个半胱氨酸残基，这是该酶钙调蛋白结合结构域的一个不寻常特征。
