• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Purification of the Constitutive Nitric Oxide Synthase

        互联网

        712
        Constitutive nitric oxide synthase (NOS) exists as two isoforms: endothelial (e) and neuronal (n) (reviewed in ref. 1 ). The first reports on the purification of eNOS and nNOS were by Schmidt et al. (2 ) and Bredt and Snyder (3 ), respectively; all subsequent reports have been modifications of these procedures. The purification of both of the constitutive isoforms and the inducible (i) NOS depends on the use of 2′,5′-adenosine diphosphate (ADP) affinity chromatography. The 2′,5′-ADP ligand is a structural mimic of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) that is required for NOS activity. Therefore, the biospecific elution is accomplished by the presence of NADPH. The purification of both constitutive isoforms, unlike iNOS, also depends on a second affinity-chromatography step, calmodulin (CaM)-affinity chromatography. Exogenously-added CaM is essential for both purified eNOS and nNOS activity. In the presence of calcium, CaM binds both constitutive isoforms in a tight (Kd ∼ 10 nM ) but reversible complex, thereby activating NOS. In contrast, CaM binds to iNOS in the absence of added calcium and dissociates only in the presence of a protein denaturant. Thus, during the 2′,5′-ADP-affinity step, CaM is removed from eNOS and nNOS, but not iNOS. The binding of eNOS and nNOS to the CaM resin is performed in the presence of calcium, and elution of NOS is achieved by the absence of calcium and presence of ethylenediaminetetraacetic acid (EDTA) and high salt. Depending on the source of enzyme, near homogenous NOS is often obtained by utilizing these two affinity steps.
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序