Kinase experiment:

NOS activity is determined by monitoring the conversion of L-[14C]arginine to [14C]citrulline. For nNOS, reaction mixtures contain, in a final volume of 200 μL, 20 mM Na+ HEPES buffer, pH 7.5, 100 μM EDTA, 2 mM CaCl2, 10 μg/mL calmodulin, 500 μM dithiothreitol, 100 μM THB, 25 μM FAD, 25 μM FMN, 100 μg/mL bovine serum albumin, 20 μM L-[14C]citrulline, 500 PM NADPH, and enzyme. Reaction is initiated by the addition of L-[14C]citrulline; reaction temperature is 25℃. At appropriate times, typically 3.5, 7, and 10.5 min, 50-1.11 portions are removed and added to 200 pl of a stopping solution of 100 mM Na+ HEPES buffer, pH 5.5, containing 5 mM EGTA. Those samples are immediately heated for 1 min in a boiling water bath, chilled, and centrifuged. A portion (225 μL) of the supernatant is fractionated on small Dowex 50 columns. [14C]citrulline is eluted with 2 ml of water and is quantitated by liquid scintillation counting[1].

L-NMMA acetate is a nitric oxide synthase inhibitor of all NOS isoforms including NOS1, NOS2, and NOS3. The Ki values for nNOS (rat), eNOS (human), and iNOS (mouse) are approximately 0.18, 0.4, and 6 μM, respectively.

L-NMMA, starting from 100 μM, produces a concentration-dependent inhibition of the evoked relaxations (2Hz); maximal inhibition at 1 mM averaged about 35%. The inhibitory effect of L-NMMA is unchanged by previous incubation with D-arginine while it is prevented by L-arginine (L-Arg). L-NMMA does not affect isoprenaline-induced relaxation[2]. Superfusion of L-NMMA reduces arteriolar diameter and causes dose-dependent increases in arteriolar tone. The onset of action of L-NMMA is nearly immediate. L-NMMA inhibits vasodilator responses to the endothelium-dependent vasodilator ACh but not to the endothelium-independent NP. NE induced dose-related vasoconstriction that is significantly potentiated by L-NMMA[3].

References:
[1]. Frey C, et al. L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases. J Biol Chem. 1994 Oct 21;269(42):26083-91.
[2]. Maggi CA, et al. Effect of NG-monomethyl L-arginine (L-NMMA) and NG-nitro L-arginine (L-NOARG) on non-adrenergic non-cholinergic relaxation in the circular muscle of the human ileum. Br J Pharmacol. 1991 Aug;103(4):1970-2.
[3]. Nakamura T, et al. Effect of NG-monomethyl-L-arginine on arcade arterioles of rat spinotrapezius muscles. Am J Physiol. 1991 Jul;261(1 Pt 2):H46-52.