Lee S J, Stull J T
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235, USA.
J Biol Chem. 1998 Oct 16;273(42):27430-7. doi: 10.1074/jbc.273.42.27430.
Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain.
神经元型和内皮型一氧化氮合酶的激活依赖于Ca2+/钙调蛋白，而诱导型一氧化氮合酶的活性不依赖于Ca2+，这可能是由于其紧密结合的钙调蛋白所致。为了研究这些不同的机制，对一系列源自神经元型和诱导型一氧化氮合酶的嵌合体进行了分析。仅包含诱导型一氧化氮合酶加氧酶结构域、钙调蛋白结合区域或还原酶结构域的嵌合体并未赋予显著的不依赖Ca2+的活性。然而，每个嵌合体对Ca2+的敏感性都高于神经元同工型。诱导型一氧化氮合酶的钙调蛋白结合区域与其加氧酶或还原酶结构域结合，导致了显著但不完全的不依赖Ca2+的活性。免疫共沉淀实验表明，在没有Ca2+的情况下，前一种嵌合体没有与钙调蛋白结合。在没有Ca2+的情况下，三氟拉嗪也抑制了这种嵌合体。与还原酶结构域相比，结合在诱导型一氧化氮合酶钙调蛋白结合区域的钙调蛋白与加氧酶结构域之间的联合相互作用可能较弱。因此，诱导型一氧化氮合酶不依赖Ca2+的活性似乎是由钙调蛋白与加氧酶和还原酶结构域以及典型的钙调蛋白结合区域的协同相互作用所导致的。神经元同工型不受其还原酶结构域中独特的自抑制元件的调节。
