Inactivation of Nitric Oxide Synthase Isoforms by Diaminoguanidine andNG-Amino-L-arginine

Wolff, Donald J.; Lubeskie, Andrew

doi:10.1006/abbi.1996.0028

Cited by 26 publications

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“…The findings for NAA are consistent with a suicide mechanism of inactivation whereby a substrate is metabolized to a reactive intermediate that covalently alters nNOS and inactivates the enzyme. These results are also consistent with those established for the NAA-mediated inactivation of nNOS purified from GH 3 pituitary cells (Wolff and Lubeskie, 1996). Because the kinetics of this reaction were reported previously (Wolff and Lubeskie, 1996), we chose to focus on the mechanism of inactivation of nNOS by NAA.…”

Section: Resultssupporting

confidence: 85%

“…Based on the reported K i values, however, nNOS (K i ϭ 0.3 M) does seem to be slightly more sensitive to inactivation by NAA compared with the endothelial (K i ϭ 2.5 M) or inducible (K i ϭ 3 M) isoforms of NOS (Wolff and Lubeskie, 1996). NAA has also been demonstrated to be highly effective against the NOS isoforms in vivo by antagonizing L-arginine-mediated endothelium-dependent relaxation with greater potency than N G -methyl-L-arginine, another metabolism-based inactivator of NOS (Fukuto et al, 1990).…”

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confidence: 94%

“…The compound N G -amino-L-arginine (NAA), which is an arginine analog that contains a hydrazine group on the guanidino moiety, has been described previously as a metabolism-based inactivator of all three NOS isoforms in vitro (Wolff and Lubeskie, 1996). Based on the reported K i values, however, nNOS (K i ϭ 0.3 M) does seem to be slightly more sensitive to inactivation by NAA compared with the endothelial (K i ϭ 2.5 M) or inducible (K i ϭ 3 M) isoforms of NOS (Wolff and Lubeskie, 1996).…”

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confidence: 99%

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Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo

et al. 2002

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It is established that NG -amino-L-arginine (NAA) is a metabolism-based inactivator of all three major nitric-oxide synthase (NOS) isoforms. The mechanism by which this inactivation occurs, however, is not well understood. In the current study, we discovered that inactivation of the neuronal isoform of NOS (nNOS) by NAA in vitro results in covalent alteration of the heme prosthetic group, in part, to products that contain an intact porphyrin ring and are either dissociable from or irreversibly bound to the protein. The alteration of the heme is concomitant with the loss of nNOS activity. Studies with nNOS containing a 14 C-labeled prosthetic heme moiety indicate that the major dissociable product and the irreversibly bound heme adduct account for 21 and 28%, respectively, of the heme that is altered. Mass spectral analysis of the major dissociable product gave a molecular ion of m/z 775.3 that is consistent with the mass of an adduct of heme and NAA minus a hydrazine group. Peptide mapping of the irreversibly bound heme adduct indicates that the heme is bound to a residue in the oxygenase domain of nNOS. We show for the first time that metabolismbased inactivation of nNOS occurs in vivo as highly similar heme products are formed. Because inactivation and alteration may trigger ubiquitination and proteasomal degradation of nNOS, NAA may be a useful biochemical tool for the study of these basic regulatory processes.

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confidence: 85%

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Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo

et al. 2002

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“…N -Methyl-L-arginine (L-NMA), the prototypic NOS inhibitor (28), has been shown, for example, to reverse the hypotension of septic shock (14,15,29) and cytokine-induced shock (17,30,31) in animal studies and in early clinical trials. Unfortunately, NMA and several other L-arginine analogs including N -amino-L-arginine (32)(33)(34) and (35,36) show little isoform selectivity; their pharmacological use may thus cause undesirable inhibition of physiological processes controlled by nontargeted NOS isoforms. We (37,38) and others (39) have shown that the S-alkyl-L-thiocitrullines show modest selectivity (up to 50-fold) for nNOS over eNOS and iNOS and have proposed that these compounds may be of use in treating dis-* This work was supported in part by National Institutes of Health grant DK48423.…”

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confidence: 99%

N5-(1-Imino-3-butenyl)-l-ornithine

Babu

Griffith

1998

Journal of Biological Chemistry

126

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Nitric oxide synthase (NOS) catalyzes the NADPHand O 2 -dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-L-NIO (N 5 -(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNOS in competition with L-arginine (K i ‫؍‬ 100 nM); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O 2 , L-VNIO irreversibly inactivates nNOS (k inact ‫؍‬ 0.078 min ؊1 ; K I ‫؍‬ 90 nM); inactivation is Ca 2؉ /calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by L-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain ϳ75% of the rate of inactivation seen with nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to date.Nitric oxide synthase (NOS) 1 catalyzes the oxidation of Larginine to nitric oxide (NO) and citrulline; NADPH and O 2 are cosubstrates (1-3). Three major isoforms of NOS have been identified to date. The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed and are regulated by Ca 2ϩ /calmodulin, whereas the activity of the inducible isoform (iNOS) is controlled transcriptionally and is not affected by changes in intracellular Ca 2ϩ . Although amino acid sequence homology among the isoforms is limited (ϳ50%) (3), all are comprised of a C-terminal reductase domain that binds NADPH and the cofactors FAD and FMN and a N-terminal oxygenase domain that binds L-arginine and the heme and tetrahydrobiopterin (BH 4 ) cofactors (1-3). The reductase domain is related in function and amino acid sequence to cytochrome P450 reductase (4), whereas the oxygenase domain is related in function (but not sequence) to the cytochromes P450. Binding of Ca 2ϩ /calmodulin to a region between the domains permits electron flow from the reductase domain to the oxygenase domain and also stimulates electron flow within the reductase domain (5). The resulting reduction of the heme cofactor allows O 2 to be activated, permitting the cytochrome P450-like oxidation of L-arginine to N -hydroxy-L-arginine and the subsequent further oxidation of that tightly bound intermediate to c...

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“…More recently, it was shown that AG is an isoform-selective mechanism-based inactivator of iNOS in vitro, although the constitutive NOS in GH 3 pituitary cells and that in endothelial cells could also be inactivated (18). Diaminoguanidine, which is structurally related to AG, is also a mechanism-based inactivator of all three isoforms of NOS, although it exhibits less selectivity for iNOS (19). The mechanism by which AG causes the inactivation of NOS is not known.…”

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Aminoguanidine-mediated Inactivation and Alteration of Neuronal Nitric-oxide Synthase

Jianmongkol

Vuletich²,

Bender³

et al. 2000

Journal of Biological Chemistry

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It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [ 14 C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.

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Inactivation of Nitric Oxide Synthase Isoforms by Diaminoguanidine andNG-Amino-L-arginine

Cited by 26 publications

References 0 publications

Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo

Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo

N5-(1-Imino-3-butenyl)-l-ornithine

Aminoguanidine-mediated Inactivation and Alteration of Neuronal Nitric-oxide Synthase

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Inactivation of Nitric Oxide Synthase Isoforms by Diaminoguanidine andNG-Amino-L-arginine

Cited by 26 publications

References 0 publications

Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byNG-Amino-l-arginine in Vitro and in Vivo

Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byNG-Amino-l-arginine in Vitro and in Vivo

N5-(1-Imino-3-butenyl)-l-ornithine

Aminoguanidine-mediated Inactivation and Alteration of Neuronal Nitric-oxide Synthase

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Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo

Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation byN^G-Amino-l-arginine in Vitro and in Vivo