Inhibition of Purified nNOS by NO

The studies discussed above were performed on unpurified preparations of the constitutive isoforms nNOS and eNOS from rat cerebellum and BAECs, respectively, as well as iNOS from activated rat alveolar macrophages. The results indicated that NO inhibits the activities of these isoforms. However, since the studies were performed the unpurified preparations, it was not possible to determine whether NO produces its inhibitory effect by a direct interaction with NOS or by an indirect mechanism involving enzymes or other factors present in the crude cellular preparations. To rule out involvement of constituents in the crude preparations, studies were undertaken with purified nNOS. Studies with purified preparations also made it possible to further elucidate the mechanism of action of NO (Griscavage et al., 1994). 

Purification of nNOS from rat cerebellum was obtained by a modification of the two-column method reported by Bredt and Snyder (1990 Griscavage et al., 1994). The procedure yielded NOS preparations that were 95% pure, with specific activities ranging from 450 to 650 nmol of L-citrulline/ min/mg of protein. Despite the presence of excess substrate and cofactors, 

The first potential site for NO binding and subsequent inhibition of NOS activity is the arginine binding site. This site could be excluded, however, because a double-reciprocal plot of substrate concentration versus reaction rate in the presence of increasing concentrations of the NO donor, SNAP (30 and 100 /xM), indicated that NO inhibited NOS by mechanisms that are not competitive with L-arginine. Likewise, SOD decreased the V ax without altering the for L-arginine. 

The heme iron in NOS exists in two distinct oxidation states Fe , or ferrous iron, and Fe , or ferric iron. Spectral studies have shown that in 

FIGURE 5 Influence of tetrahydrobiopterin (H4B) on the inhibitory action of nitric oxide (NO) on nNOS activity. Standard enzyme incubations were conducted for 10 min as described previously (Griscavage et al., 1994) and are similar to the conditions in Fig. 1. NO was added to reaction mixtures immediately after addition of NOS. H4B was added to reaction mixtures just prior to initiation of reactions by addition of NOS. Control reaction mixtures contained 10 fiM H4B. Data represent the means se of duplicate determinations from four separate experiments. 

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