Premium
Absence of Nitric Oxide Synthase in Exquisitely Pure Rat Liver Mitochondria
Author(s) -
Venkatakrishnan Priya,
Nakayasu Ernesto S.,
Almeida Igor C.,
Miller Richard T.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.835.3
Subject(s) - gene isoform , mitochondrion , citrulline , nitric oxide synthase , nitric oxide , percoll , arginine , biochemistry , differential centrifugation , western blot , centrifugation , biology , microbiology and biotechnology , chemistry , enzyme , endocrinology , amino acid , gene
The nitric oxide synthases (NOS) catalyze the conversion of L ‐arginine to L ‐citrulline plus NO. Data, both for and against the presence of a mitochondrial NOS (mtNOS) isoform, is in the refereed literature base. Irrefutable evidence, however, for a mtNOS isoform has not been forthcoming and conflicting reports continue to be published. In light of this controversy, we designed meticulous studies to confirm/refute the existence of mtNOS. Pure, rat liver mitochondria were obtained by differential centrifugation followed by Percoll purification. Positive controls contained mitochondria spiked with purified, recombinant nNOS (nNOSr). Analysis of sub‐mitochondrial particles by LC‐MS failed to detect any NOS isoform in mitochondria whereas nNOSr peptides in spiked samples as low as 50 fmol were detected. [C 14 ] L ‐arginine to [C 14 ] L ‐citrulline conversion was negative for NOS activity. Additionally, detection of NO production by mitochondria by use of the oxyhemoglobin capture assay run with or without SOD & catalase were also negative. Moreover, Western blot analyses using nNOS & eNOS antibodies failed to detect these two isoforms in mitochondria. In conclusion, by avoiding previously published errors in methodologies and sample preparation, and by using correct controls, we refute the claim that a NOS isoform exists within rat liver mitochondria. (Supported by ES011982 & RR008124 to RTM & UTEP, respectively)