Therefore, by exclusion, it appears that the 320kD band represented nNOS. bands when blotted with anti-nNOS1422C1433 and 320 and 155kD bands when blotted with anti-nNOS1C20 antibodies respectively. The 320kD and 155kD bands represent dimers and monomers of nNOS; the 250kD and 135kD bands represent dimers and monomers of nNOS. Immunoprecipitation with calmodulin (CaM) showed that a portion of nNOS dimer was bound with CaM. On the other hand, a portion of nNOS dimer, nNOS dimer and all monomers lacked CaM binding. The CaM-lacking nNOS fractions reacted with anti-serine847-phospho-nNOS. assays of NO production revealed that only the CaM-bound dimeric nNOS was catalytically active; all other forms were inactive. We suggest that the amount of catalytically active nNOS dimers may be regulated by serine847 phosphorylation and equilibrium between dimers and monomers of nNOS. assay system. MATERIALS AND METHODS The experimental protocols used in RS 127445 this study were approved by the Institutional Animal Care and Use Committee of VA Boston Healthcare System. Antibodies and Chemicals Details of all the antibodies used in the present study are summarized in table 1. The anti-ser847-phospho-nNOS antibody was raised against 848KRFNSVS854 of human nNOS that recognized serine 852 phosphorylation in the human sequence and corresponded with the sequence 844RFNSVS849 in the mouse nNOS (Santa Cruz). Thus, in mice, this antibody recognizes the serine847-phosphorylated nNOS. All secondary antibodies were obtained from Jackson Immunochemicals and Santa Cruz. Reagents for immunoblot and chemicals used were from BioRad, Wako Chemicals and Sigma. Immunoprecipitation reagents (Protein A/G) were from Roche Molecular Biochemicals and Santa Cruz. Table 1 Details of antibodies used in the current study. assay (49.19 1.47 picomole/30min/mg protein). This nitric oxide production was significantly suppressed after pharmacologic treatment with L-NAME (p 0.05), a drug that inhibits all forms of nNOS enzyme. The nitric oxide production was also significantly suppressed by 7-nitroindazole (p 0.05), a drug that inhibits nNOS enzyme by inhibiting BH4 and consequently disrupting nNOS dimer formation. Nitric oxide was not produced when L-arginine was removed from the assay mixture. These studies show that CaM-associated nNOS was catalytically active. In Ace contrast, precipitates of varicosity extracts with serine-847-phosphorylated-nNOS antibodies (i.e., serine-phosphorylated nNOS) RS 127445 showed very low levels of NO production (9.60 1.45 pMole/30min/mg protein). The nNOS inhibitors L-NAME and 7-NI did not further inhibit NO production in these samples (p 0.05). Additionally, the floating fraction obtained after CaM-IP of the varicosity extract showed scant or no production of nitric oxide in the assay, showing that the CaM-lacking nNOS in the supernatant could not generate nitric oxide. These data indicate that serine-847-phosphorylated nNOS was catalytically inactive. Heat treated varicosity samples (37 C) did not show any detectable level of NO during the functional assay, suggesting that monomers of nNOS were ineffective in NO synthesis. Open in a separate window Figure 6 Quantification of nitric oxide production by different nNOS fractions in mice nerve terminals NO synthesis as assessed by a fluorimetric assay using DAF-2 in whole varicosity extracts, calmodulin IP and phospho-serine-847-nNOS IP samples. For each of the categories, the nitric oxide assays were performed in quadruplicates. A nitric oxide donor, nor-3, was used to calculate the standard graph from which nitric oxide production in unknown biological samples were computed (shown on the top). Note the robust production of nitric oxide by the CaM-IPs as compared to ser847-P-nNOS-IPs. Also note that heat (37 C) and omission of L-arginine from the samples resulted in total absence of NO synthesis. DISCUSSION This study shows that in the enteric varicosity extracts: 1) nNOS was present as 320kD, 250kD, 155kD and 135 kD bands; 2) These bands represented dimers and monomers of and isoforms of nNOS RS 127445 respectively; 3) 320kD nNOS dimer existed in both CaM-bound and CaM-free forms; 4) CaM-associated nNOS dimer was catalytically active, while the CaM-lacking nNOS dimer was catalytically inactive and was phosphorylated at serine 847; 5) 155kD nNOS monomer and 250kD RS 127445 nNOS dimers lacked CaM and were also catalytically inactive. Various isoforms of nNOS, namely , , , and nNOS2 isoforms, are produced by the post-transcriptional splicing of nNOS mRNA (22, 23). The dimers of nNOS a, , , and nNOS2 are known to have approximate molecular weights.