An assay method for nitric oxide synthase in crude samples by determining product NADP

An assay method for nitric oxide synthase (NOS) was developed based on fluorometric or enzymatic determination of NADP+. An aliquot (< or = 2 microliters) of crude enzyme sample, homogenate or supernatant of rat cerebellum, was added to a reaction mixture containing arginine, NADPH, and O2 and incubated at 25 degrees C for 30 min. A strongly fluorescent substance was formed from a product, NADP+, and measured (fluorometric assay). When rat cerebellar layers were assayed, freeze-dried sections of layers (0.2 to 2 micrograms dry wt) were added directly into 1.24 microliters of NOS reaction mixture and the total NADP+ formed in picomole amounts was specifically amplified up to 4000-fold and determined, using an enzymatic NADP cycling amplification reaction (enzymatic-cycling assay). NOS activity was calculated as the difference in NADP+-forming activity in the absence and presence in the reaction mixture of NG-nitro-L-arginine, a specific inhibitor of NOS. Enzymatic activity was analyzed in rat cerebellar supernatants by two procedures. With supernatants (and purified macrophage NOS), the ratio between the specific activities on a protein basis using the present NADP+ formation assay and using [3H]citrulline formation from [3H]arginine as substrate was 2. The distribution of NOS activity was shown between the particulate and supernatant fractions of rat cerebellum. The molecular and granular layers of rat cerebellum contained similar NOS activities, while the activity in the white matter was negligibly low. NOS distribution is also reported among rat organs.