FAD and GSH participate in macrophage synthesis of nitric oxide.
FAD and GSH participate in macrophage synthesis of nitric oxide.
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DOI：
10.1016/0006-291x(90)92357-6
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发表时间：
1990-04
影响因子：
3.1
通讯作者：
D. Stuehr;Nyoun Soo Kwon;Carl Nathan
中科院分区：
文献类型：
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作者：
D. Stuehr;Nyoun Soo Kwon;Carl Nathan
巨噬细胞一氧化氮（NO）合成酶部分纯化后，酶活性需要l -精氨酸、NADPH和组成型胞浆因子，其中一种是四氢生物蝶呤（BH4） （Kwon， N.S, Nathan， C.F., Stuehr， D.J. 1989）。化学。264,20496)。在这里，我们确定FAD和谷胱甘肽作为两个额外的辅助因子需要充分的酶活性。在所有确定的细胞质辅助因子过量的情况下，NO的合成在3小时内呈线性，并且约50%依赖于外源性FAD，约50%依赖于谷胱甘肽（GSH）， 84%依赖于四氢生物terin (BH4)， 95%依赖于NADPH， 98%依赖于l -精氨酸。最佳活性所需的添加FAD、GSH和bh4的浓度与其在巨噬细胞细胞质中的水平一致。动力学研究表明，谷胱甘肽（或DTT）在反应的前20-30分钟内对no生成速率几乎没有影响，但阻止了随后速率的下降。这种作用不同于硫醇参与bh4再生。相比之下，外源性FAD在整个实验期间使NO合成速率增加了一倍，这与辅助因子的作用一致。NADPH的作用不是再生BH4，提供NADP+，也不是形成活性氧中间体。这些发现表明，在另一个确定的系统中，部分纯化的酶可以合成NO，并表明利用nadph的FAD黄蛋白可能参与了该反应。
Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was ∼50% dependent on exogenous FAD, ∼50% on glutahione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20–30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.