title: Alteration of the heme prosthetic group of neuronal nitric-oxide synthase during inactivation by N(G)-amino-L-arginine in vitro and in vivo.
abstract: It is established that N(G)-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 14C-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 metabolism-based 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.
authors.affiliation: Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Vuletich
authors.forename: Ezra R
authors.initials: ER
authors.lastname: Lowe
authors.forename: Suree
authors.initials: S
authors.lastname: Jianmongkol
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kamada
authors.forename: Ute M
authors.initials: UM
authors.lastname: Kent
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Bender
authors.forename: Damon R
authors.initials: DR
authors.lastname: Demady
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Osawa
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D006418:Heme; C063904:N(G)-aminoarginine; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat