Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.
Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.
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Ca2/钙调蛋白依赖性 NO 合酶 I 型：一种生物蝶黄蛋白，具有不依赖 Ca2/钙调蛋白的心肌黄酶和还原酶活性。
DOI：
10.1021/bi00127a028
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Murad,F
中科院分区：
文献类型：
--
作者：
Schmidt,HH;Smith,RM;Nakane,M;Murad,F
1992年1月16日收到的修订手稿摘要：没有合成酶(NOS；EC 1.14.23)催化L-精氨酸转化为L-瓜氨酸和鸟苷酸环化酶激活因子，在化学上与一氧化氮或一氧化氮释放化合物(NO)相同。与迄今已确定的其他一氧化氮合酶同工酶相似，大鼠小脑的可溶型和钙/钙调蛋白调节的I型(160 kDa亚基的同源二聚体)依赖于NADPH的催化活性。该酶在电子受体硝基蓝四氮唑(NBT)存在下也具有NADPH黄递酶活性。我们调查了所建议的附加辅因子四氢生物蝶呤和黄素对一氧化氮合酶的需求及其含量，进一步表征了NADPH黄递酶的活性，并量化了NADPH结合位点(S)。纯化的NOS I型钙/钙调素独立地与NADPH的[32P]2/，3/-双醛类似物(DNADPH)结合，以接近KM浓度的dNADPH为底物，在较高浓度或长时间孵育和交联后抑制NOS活性。NADPH黄递酶活性不依赖于钙/钙调蛋白，需要比NOS活性更高的NADPH浓度，受dNADPH影响较小。二价阳离子干扰黄递酶测定。在每个二聚体中，天然的NOS分别含有约1摩尔的H4生物蝶呤、FAD和FMN，将其归类为生物蝶呤蛋白，并加入1摩尔的dNADPH。未检测到二氢生物蝶呤、生物蝶呤或核黄素。这些发现表明，一氧化氮合酶可能通过高亲和力结合位点在两个相同的亚基之间共享辅因子。他们还解释了为什么不同的NOS制剂对外源黄素和H4生物蝶呤有不同的要求，以达到最大的催化活性。此外，单独使用H4生物蝶呤可降低NBT，纯化的二氢蝶啶还原酶具有黄递酶活性。除了H4生物蝶呤以外，没有酶结合的生物蝶呤，甲氨蝶呤对酶活性的影响缺乏报道，以及NBT对NOS活性的有效和非竞争性抑制，这也表明NOSI型可能具有一种新的甲氨蝶呤不敏感的奎宁-H_2生物蝶呤还原酶活性。
Revised Manuscript Received January 16, 1992 abstract: NO synthase (NOS; EC 1.14. 23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical withnitric oxide or a nitric ox-ide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in thepresence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site (s). Purified NOS type I Ca2+/calmodulin-independently boundthe [32P] 2/, 3/-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linkinginhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS mayshare cofactors between two identical subunits via high-affinity binding sites. They also explain why different preparations of NOS have different requirements for exogenous flavins and H4biopterin for maximal catalytic activity. Furthermore, H4biopterin alone reduced NBT, and purified dihydropteridine reductase was found to have diaphorase activity. The absence of enzyme-bound biopterins other than H4biopterin, the reported lack of effect of methotrexate on enzyme activity, and the potent and noncompetitive inhibition of NOS activity by NBT suggest also that NOS type I mayhave a novel methotrexate-insensitivequinoid-H2biopterin reductase activity.