CLONED, EXPRESSED RAT CEREBELLAR NITRIC-OXIDE SYNTHASE CONTAINS STOICHIOMETRIC AMOUNTS OF HEME, WHICH BINDS CARBON-MONOXIDE
CLONED, EXPRESSED RAT CEREBELLAR NITRIC-OXIDE SYNTHASE CONTAINS STOICHIOMETRIC AMOUNTS OF HEME, WHICH BINDS CARBON-MONOXIDE
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DOI：
10.1073/pnas.89.23.11141
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发表时间：
1992-12-01
影响因子：
11.1
通讯作者：
MASTERS, BSS
中科院分区：
文献类型：
--
作者：
MCMILLAN, K;BREDT, DS;MASTERS, BSS
一氧化氮（NO）的内源性形成已成为一个非常感兴趣的领域，因为其生物学功能的证据已在三种不同的组织中获得：循环巨噬细胞，其中它发挥细胞毒性作用;血管，其中它已被鉴定为内皮衍生的松弛因子;和神经元细胞，其中它作为神经递质发挥作用。脑提取物中NO的形成已被证明是由称为NO合酶的酶催化的，该酶产生负责刺激cGMP形成的NO，其最高水平发生在小脑中。NO合酶催化精氨酸形成瓜氨酸，同时产生NO，并且已被证明是黄素蛋白，含有1 mol FAD和FMN、四氢生物蝶呤和铁。据报道，它还含有一个α-螺旋，钙调蛋白结合的共识序列与其刺激钙调蛋白在Ca 2+的存在下。NO的形成需要将一个分子氧原子结合到精氨酸的一个胍氮原子中，同时形成瓜氨酸。该通讯报道，大鼠小脑NO合酶，克隆和稳定表达在人肾293细胞，含有血红素的量与黄素FAD和FMN的化学计量证明了吡啶血色素和减少CO差光谱的外观与吸收最大值几乎等于445 nm。一氧化碳结合血红素部分的发现解释了铁在酶中的存在，并建议假体血红素作为加氧酶反应中心的作用。本报告还提供了证据，纳入δ-[C-14]aminolevulinate特异性免疫沉淀NO合成酶在稳定转染的人肾293细胞，但不是在非转染细胞。同时，K。A.白色和M. A. Marletta [（1992）Biochemistry 31，6627-6631]已经证明在纯化的鼠巨噬细胞NO合酶中存在CO-结合血红素辅基，并且已经提出在NO合酶的组成型（小脑）和诱导型（巨噬细胞）形式中这些反应中心的同一性。
The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at almost-equal-to 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[C-14]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in purified murine macrophage NO synthase and have suggested the identity of these reaction centers in both the constitutive (cerebellar) and inducible (macrophage) forms of NO synthase.