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Fig. 7. NO formation from nitrite reduction by eNOS and nNOS at 500 pM nitrite. The reaction is initiated by injection of 100 pM of NADPH.The electrode current and optical density are studied simultaneously in a single experiment. (A) NO release as monitored by the NO electrode current. Upper trace is 5 pM eNOS. Lower trace is 7 pM nNOS. (B) Formation of ferrous heme-nitrosyl complex after injection of NADPH. Triangles represent 5 pM eNOS, the squares 7 pM nNOS. The right scale gives the degree of nitrosylation as computed from AE442 = [£442 (NO — heme) — £442 (heme)] = 4 x 104 M—1 cm 1. The experiments are performed under a constant argon flow in 50 mM Tris buffer at pH = 7.6 containing 150 mM NaCl and 5% glycerol, 1 mM Ca2+.

1 mM arginine, 40 pM BH4, 10 pM calmodulin,

(Fig. 7A) showed that the NO was released from the enzyme into the solution. In contrast, the electrode current (Fig. 7A) showed that anoxic nNOS failed to release significant quantities of free NO. When the measurement of nNOS was continued for 90 min, a small signal corresponding to 0.32 ± 0.07 ^M NO could be observed.

Fig. 7B shows the change in absorbance at 442 nm, simultaneously recorded from the same solution, which is indicative of heme nitrosylation. The kinetics of Fig. 7A shows that free NO was released in a fast burst within a few seconds required for mixing NADPH. This release of NO is clearly faster than the nitrosylation of eNOS heme, attesting some NO rebinding to the protein after being released.

It is interesting to note that the eNOS and nNOS exhibit significantly different degrees of nitrosylation (cf Fig. 7 and Table 3). The optical density at 442 nm showed that the degree of nitrosylation of eNOS reached 9% after 5 min and increased to 18% after 25 min. For nNOS the numbers are far higher with 25% at 5 min, 55% at 25 min, and seemed to saturate at an asymptotic value of 62% nitrosylation.

Although the electrode detected the release of small quantities of free NO, the extensive nitrosylation of nNOS shows that the dominant fraction of NO is retained as a nitro-syl ligand by nNOS. Clearly, the NO was formed but was unable to diffuse out of the protein.

Table 3 Percentage of nitrosylated heme at given time points after injection of NADPH. Data are taken from Fig. 7

Table 3 Percentage of nitrosylated heme at given time points after injection of NADPH. Data are taken from Fig. 7

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