Table 1.

Activity of Purified Recombinant O. tauri NOS

Conditions	NOS	NO Formation (min−1)	%	Citrulline Formation (min−1)	%
Cofactors omitted
None	O. tauri NOS	0.49 ± 0.03	100	0.53 ± 0.05	100
CaM	O. tauri NOS	0.35 ± 0.01	71	0.37 ± 0.01	69
H4B	O. tauri NOS	<0.001	<1.0	<0.001	<1.0
Addition of inhibitora
l-NAME	O. tauri NOS	0.10 ± 0.10	20	<0.001	<1.0
Cofactors omitted
None	iNOS	0.59 ± 0.13	120	0.41 ± 0.14	77

NO production was determined by the oxyhemoglobin method. Activity was measured for 3 min at 25°C in reaction containing 20 μM oxyhemoglobin, 5 mM DTT, 100 μM l-Arg, 1 mM NADPH, 10 mM CaCl₂, 10 μM CaM, 100 μM H₄B, 100 units/mL catalase, and 0.5 μM O. tauri NOS or commercial iNOS (Sigma-Aldrich). [³H]l-citrulline formation was measured for 30 min in a reaction containing 1 μCi [³H] l-Arg, 50 μM unlabeled l-Arg, 100 μM NADPH, 10 μM FAD, 2 mM CaCl₂, 1 μg CaM, 100 μM H₄B, and 0.5 μM O. tauri NOS or commercial iNOS. l-NAME was used at concentration 100 μM. Activity assays were performed without adding FAD and FMN. The maximal activity was obtained without adding FAD and FMN, indicating that recombinant O. tauri NOS is replete with FAD and FMN as was previously described for other NOS recombinant activities (Martasek et al., 1996).

^aActivity assay in presence of all cofactors.