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 expres sed in the baculovirus system and purified by a novel immunoaffinity c olumn. rH-iNOS and its native counterpart from cytokine-stimulated pri mary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-po lyacrylamide gel electrophoresis, recognition by antipeptide antibodie s, specific activities, and IC50 values for inhibitors. The active dim eric form exhibited a specific activity range of 114-260 nmol/min/mg a t 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 reducti on under CO yielded an absorbance maximum at 446 nm, indicating a P-45 0-type heme. Imidazole induced a type II difference spectrum, reversib le 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-fer rous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduc t directly, but it partially reversed the ADT-induced absorbance shift , indicating that both bind similarly to the protein but interact diff erently with the heme.