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

Suree Jianmongkol,Jennifer L Vuletich,Andrew T Bender,Damon R Demady,Yoichi Osawa

doi:10.1074/jbc.275.18.13370

Suree Jianmongkol, Jennifer L Vuletich + Show 3 more

Open Access

https://doi.org/10.1074/jbc.275.18.13370

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Abstract

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.

Highlights

Nitric-oxide synthases (NOS)1 are cytochrome P-450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide by a process that requires NADPH and molecular oxygen [1,2,3,4]
The inactivation caused by AG was dependent on the presence of calmodulin (Fig. 1D, compare closed square with closed diamond), which is necessary for neuronal NOS (nNOS) activity, and indicates a metabolism-dependent inhibition process
The reason for the greater than 10-fold difference in the Ki is not known, IC50 values of 41 ␮M have been reported for the AG-mediated inhibition of nNOS found in GH3 pituitary cells [28]

Summary

Introduction

Nitric-oxide synthases (NOS) are cytochrome P-450-like hemoprotein enzymes that catalyze the conversion of L-arginine to citrulline and nitric oxide by a process that requires NADPH and molecular oxygen [1,2,3,4]. Similar to the findings on iNOS, the inactivation of nNOS by AG has been found to occur without changes in the heme fluorescence and to lead to irreversible binding of [14C]AG to the protein [21] It appears that AG can alter the heme or protein of NOS, but the role for either of these processes in the inactivation has not been demonstrated. Inactivation of nNOS with [14C]AG gave radiolabel associated with the dissociable heme product as well as radiolabel irreversibly bound to the protein primarily at a site(s) on the reductase domain of nNOS These studies further our understanding of the mechanisms involved in the inactivation of nNOS and may aid in predicting the action of NOS inhibitors in vivo

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