A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon y and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NOj plus NOj per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 FM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of -250 kDa but could be dissociated into inactive monomers of -130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.The free radical nitric oxide (NO) or a NO-releasing product is synthesized within mammalian immune, cardiovascular, and neural systems, where it functions as a signaling or cytotoxic molecule (for reviews, see refs. 1-3). The mammalian enzymes that generate NO are not completely characterized. Current evidence suggests that there are at least two forms. One is constitutively expressed, requires calcium ions and a calcium-binding protein such as calmodulin for its activation, and participates in signal transduction by generating NO in response to increased intracellular calcium levels, leading to activation of soluble guanylyl cyclase by NO (1, 4-7). The other form is expressed in cells only after several hours of exposure to cytokines like interferon y (IFN-y) and/or microbial products such as bacterial lipopolysaccharide (LPS) (8)(9)(10). Immunologically induced NO synthase participates in the destruction of microbial pathogens and tumor cells and contributes to shock associated with sepsis (2, 3, 11, 12). The inducible NO synthase appears to be antigenically distinct from constitutive NO synthase (13).Despite their differences in regulation and function, evidence suggests that the constitutive and induced NO synthases are catalytically similar. Both types utilize NADPH and, where it was tested, tetrahydrobiopterin as redox cofactors (4-7, 14, 15) and convert L-arginine to NO and L-citrulline with one atom of molecular oxygen being incorporated into L-citrulline (16). In cases where it was tested, both constitutive and inducible activities were enhanced by exogenous FAD (7, 17) and inhibited irreversibly by the flavoprotein inhibitor diphenyleneiodonium (18), suggesting that NO synthases may be flavoproteins. Howev...

The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.).

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SummaryMacrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting t-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N'~-methyl+arginine, or N'~-amino+arginine. t-Arginine, but not D-arginine, abolished the protective effect of N'~ The prototypic pharmacologic inhibitor of IkR is hydroxyurea. Its structural resemblance to N '~-hydroxy-t-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H202 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3-generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating reactant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.

Brains from subjects who have Alzheimer's disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human β-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS+/+ or iNOS−/−, and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased β-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of β-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B. Nitric oxide (NO)1 is produced by many cells and plays important roles in the nervous, muscular, cardiovascular, and immune systems (1-5). Neuronal NOS (nNOS, NOS 1) and endothelial NOS (eNOS, NOS 3) produce low NO concentrations for neurotransmission, insulin release, penile erection, vasorelaxation, oxygen detection, and memory storage, whereas cytokine-inducible NOS (iNOS, NOS 2) produces larger NO concentrations to counter pathogens and coordinate the immune response. The iNOS binds calmodulin (CaM) very tightly and is not regulated by changes in intracellular Ca 2ϩ , whereas the eNOS and nNOS isoforms both bind CaM reversibly and are regulated by intracellular Ca 2ϩ (6 -8). All three NOS exhibit a similar catalytic profile and composition. They catalyze a two-step oxidation of L-arginine (Arg) to form NO and citrulline, with N-hydroxy-L-arginine (NOHA) being formed as an enzyme-bound intermediate (9 -11). The amino-terminal portion of each NOS represents an oxygenase domain (amino acids 1-498 in mouse iNOS) that binds Feprotoporphyrin IX (heme), Arg, and (6R)-tetrahydrobiopterin (H4B) (12, 13). The carboxyl-terminal portion of NOS represents a reductase domain (amino acids 533-1172 in mouse iNOS) that contains binding sites for NADPH, FAD, and FMN and bears much resemblance to cytochrome P450 r...

Streptozotocin (STZ), a diabetogenic agent, is thought to damage pancreatic beta-cells by activating immune mechanisms and by alkylating DNA. In the present study, we demonstrated that STZ can produce nitric oxide (NO), a bioregulatory and cytotoxic molecule. When STZ was dissolved in a sodium phosphate buffer (50 mM, pH 7.4) and irradiated with a 22 W circular fluorescent light, nitrite and nitrate, stable oxidation products of NO, were produced. The wavelengths of light most responsible for the photo-decomposition were 300-310 nm and 410-420 nm. When a mixture of reduced hemoglobin and STZ was irradiated with UV light (280-320 nm), hemoglobin underwent characteristic NO-dependent spectral changes. STZ relaxed deendothelialized aortic strips only in the presence of light. STZ/light-dependent relaxation was attenuated by reduced hemoglobin. These results indicated photo-induced NO production from STZ. NO generation depended on the concentration of STZ, the duration of irradiation, and the distance between sample and light source. In acidic conditions, NO production from STZ was spontaneous even in the dark. Light-independent NO generation was augmented by increasing acidity, and markedly diminished in a D2O-based buffer, indicating the involvement of protons in the mechanism of STZ decomposition in acid. These results imply the usefulness of STZ as an NO-generating reagent, and indicate that direct NO-generation may be a mechanism of STZ toxicity in diabetogenesis.

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