Human RBCs contain an active NOS3. (A) Constitutive NOS (135 kDa) was isolated from human RBCs by affinity chromatography with a calmodulin (CaM)–binding column followed by immunoprecipitation (IP) with a mouse anti–human NOS3 or directly by immunoprecipitation from crude RBC lysates (lanes 1 and 2, as well as 3 and 4 are independent samples). (B) The NOS enzyme was isolated directly from crude red cell lysates by immunoprecipitation with a mouse anti–human eNOS antibody and analyzed by Western blotting. As a control a crude EC lysate was loaded in lane 5 (MWM indicates molecular weight marker). (C) Coomassie gel used for identification purposes and representative example of peptide identification by LC-MS/MS sequencing; depicted is the fragmentation spectrum of the peptide AQSYAQQLGR. (D) Alignment of peptides identified by LC-MS/MS with sequences of nNOS, iNOS and all known eNOS splice variants. Top panel: Peptides 1 and 2 aligned within the FMN reductase-like region (FMN binding region), whereas peptides 3 and 4 aligned with the ferredoxin reductase (FNR)–like region (FAD and NADPH binding region). CysJ indicates sulfite reductase, α subunit (flavoprotein) region. NOS oxygenase indicates nitric oxide synthase eukaryotic oxygenase domain. Bottom panels: The sequences shown in detail and compared with the other NOS isoforms. The sequences of the peptides are identical with NOS3, isoform 1 only. NP_000611 = NOS1, nNOS, NP000616 = NOS2, iNOS, NP_000594 = NOS3, isoform 1. Splice variants of NOS3 are NP_001153581 = NOS3, isoform 2, NP_001153582 = NOS3, isoform 3, NP_001153583 = NOS3, isoform 4. (E) Activity of immunoprecipitated red cell eNOS. The activity of NOS3 immunoprecipitated from RBCs was assessed by measuring the conversion of L-³H-arginine to L-³H-citrulline. Total radioactivity is expressed as counts per minute (cpm). (ANOVA P = .0447; Tukey IP versus IP+L-NAME *P < .05; #IP versus Ca²⁺/CaM t test P = .0394).