Expression and immunoaffinity purification of human inducible nitric-oxide synthase - Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmedulin/monomer. The enzyme exhibited a Soret lambda(max) at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 mel of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P-450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (K-i = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (K-d = 17.7 nM), and altered the GO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.