Schmidt H H, Smith R M, Nakane M, Murad F
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Biochemistry. 1992 Mar 31;31(12):3243-9. doi: 10.1021/bi00127a028.
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 with nitric oxide or a nitric oxide-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 the presence 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 bound the [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-linking inhibited 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 may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)
一氧化氮合酶（NOS；EC 1.14.23）催化L-精氨酸转化为L-瓜氨酸和一种与一氧化氮或一氧化氮释放化合物（NO）化学性质相同的鸟苷酸环化酶激活因子（GAF）。与迄今为止已被鉴定的其他NOS同工酶类似，大鼠小脑的可溶性且受Ca2+/钙调蛋白调节的I型（160 kDa亚基的同二聚体）催化活性依赖于NADPH。在电子受体硝基蓝四唑（NBT）存在的情况下，该酶还具有NADPH黄递酶活性。我们研究了NOS的需求及其对所提出的额外辅因子四氢生物蝶呤（H4biopterin）和黄素的含量，进一步表征了NADPH黄递酶活性，并对NADPH结合位点进行了定量。纯化的I型Ca2+/钙调蛋白非依赖性NOS与NADPH的[32P]2',3'-二醛类似物（dNADPH）结合，在3分钟孵育期间，接近Km浓度的dNADPH被用作底物，而在更高浓度或长时间孵育及交联后会抑制NOS活性。NADPH黄递酶活性不依赖于Ca2+/钙调蛋白，所需的NADPH浓度高于NOS活性，且受dNADPH的影响较小。二价阳离子干扰黄递酶测定。每个二聚体的天然NOS含有约1摩尔的H4biopterin、FAD和FMN，将其归类为生物蝶呤黄素蛋白，并结合1摩尔的dNADPH。未检测到二氢生物蝶呤（H2biopterin）、生物蝶呤或核黄素。这些发现表明，NOS可能通过高亲和力结合位点在两个相同亚基之间共享辅因子。（摘要截短于250字）
