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首页> 外文期刊>Biochemistry >INTERACTION OF CALMODULIN WITH THE INDUCIBLE MURINE MACROPHAGE NITRIC OXIDE SYNTHASE
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INTERACTION OF CALMODULIN WITH THE INDUCIBLE MURINE MACROPHAGE NITRIC OXIDE SYNTHASE

机译：钙调蛋白与难治性小鼠巨噬细胞一氧化氮合酶的相互作用

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The inducible isoform of nitric oxide synthase (iNOS) isolated from murine macrophages copurifies with calmodulin (CaM) as a tightly bound subunit, The exact function of this tightly bound CaM, however, has not been elucidated. In order to probe the function of this unusually strong interaction between iNOS and CaM, a 30 amino acid peptide derived from the putative CaM binding site of iNOS was synthesized. Cross-linking and autoradiographic analyses demonstrated that the peptide and CaM form a 1:1 complex as well as several higher molecular weight complexes. When assayed in the presence of a 12-fold excess of peptide to iNOS, over 90% of the enzymatic activity was inhibited. This inhibition could be prevented with the addition of exogenous bovine CaM to the assay mixture, in a concentration-dependent manner. Native PAGE and Western blot analysis of iNOS treated with peptide revealed the formation of a peptide-CaM complex with CaM derived from iNOS. Moreover, EGTA (5 mM) caused a 30% maximal inhibition of activity that was reversed by the addition of exogenous Ca2+ in a concentrationdependent fashion, suggesting a role for Ca2+ in this interaction. EGTA also changed the native PAGE mobility of iNOS and increased the intensity of a band which comigrates with CaM. These results demonstrate that the binding interaction between CaM and iNOS is tight but reversible, requires Ca2+, and is a typical from other known CaM-enzyme interactions.
机译：从鼠巨噬细胞中分离出的可诱导型一氧化氮合酶（iNOS）与钙调蛋白（CaM）作为一个紧密结合的亚基共纯化，但是尚未阐明这种紧密结合的CaM的确切功能。为了探测iNOS和CaM之间这种异常强的相互作用的功能，合成了一个30个氨基酸的肽，这些肽衍生自iNOS的假定CaM结合位点。交联和放射自显影分析表明，肽和CaM形成1：1的复合物以及几种更高分子量的复合物。当在iNOS的12倍过量的肽存在下进行分析时，超过90％的酶活性被抑制。可以通过以浓度依赖的方式向测定混合物中添加外源牛CaM来防止这种抑制作用。用肽处理的iNOS的天然PAGE和Western印迹分析显示与iNOS衍生的CaM形成了肽-CaM复合物。此外，EGTA（5 mM）导致最大30％的活性抑制作用，该抑制作用可通过以浓度依赖性方式添加外源Ca2 +来逆转，表明Ca2 +在这种相互作用中的作用。 EGTA还改变了iNOS的天然PAGE迁移率，并增加了与CaM迁移的条带强度。这些结果表明，CaM和iNOS之间的结合相互作用紧密但可逆，需要Ca2 +，并且是其他已知的CaM-酶相互作用的典型现象。

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