Table 1.

Effect of cofactors, l-arginine, and analogs of l-arginine on NADPH oxidation, l-citrulline, and DEPMPO-OOH formation by eNOS

Experiment	Enzyme		Addition	NADPH oxidation, nmol⋅min−1 per mg protein	[14C]Citrulline, nmol⋅min−1 per mg protein	DEPMPO-OOH, μM
A	eNOS		—*	50.0  ± 5.3	—†	1.5 ± 1.5
	+Ca2+/CaM	+	None	147.0  ± 6.0	—†	6.7 ± 0.3
	+Ca2+/CaM	+	1 mM l-Arg	ND	—†	6.5
	+Ca2+/CaM	+	10 μM BH4	139.5	—†	4.2
	+Ca2+/CaM	+	l-Arg + BH4¶	288.7	133.3	0.0
	+Ca2+/CaM	+	0.1 mM l-NAME	107.1  ± 3.4	—†	5.2
	+Ca2+/CaM	+	1 mM CN−	49.0	—†	<1.0
B	eNOS/+ BH4‡		—*	47.0	—†	<1.0
	+Ca2+/CaM	+	None	122.2	—†	2.5
C	eNOS		—*	50.0  ± 5.3	—†	1.5 ± 1.5
	+Flavins§	+	None	ND	—†	7.2
	+Flavins§	+	Ca2+/CaM	334.5  ± 34	—†	13.6
	+Flavins§	+	Ca2+/CaM l-Arg + BH4¶	564.3  ± 44.3	138.2 ± 9.1	1.9
	+Flavins§	+	0.1 mM DPI	1.0	—†	0.0

Values represent mean ± SD (n = 3), where appropriate. ND, not determined; DPI, diphenyleneiodonium chloride. 

^*Enzyme was incubated with NADPH in 50 mM Hepes buffer (pH 7.4) containing 0.1 mM DTPA. 

^†Assay does not contain either substrate or cofactor necessary for activity. 

^‡Enzyme isolated in the presence of BH₄. 

^§FAD (1 μM) and FMN (1 μM). 

^¶l-Arg (0.1 mM) and BH₄ (10 μM).