CHARACTERIZATION OF BOVINE ENDOTHELIAL NITRIC-OXIDE SYNTHASE AS A HOMODIMER WITH DOWN-REGULATED UNCOUPLED NADPH OXIDASE ACTIVITY - TETRAHYDROBIOPTERIN BINDING-KINETICS AND ROLE OF HEME IN DIMERIZATION

The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mu tant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexp ression system. The purified protein was soluble and highly active (0. 2-0.7 mu mol of L-citrulline mg(-1). min(-1)), contained 0.77 +/- 0.01 equivalent of haem per subunit, showed a Sorer maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of L-arginine or tetrahydrobiopterin. Radioligand binding studies reveale d K-D values of 147 +/- 24.1 nM and 52 +/- 9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM L-argini ne respectively. The positive co-operative effect of L-arginine was du e to a pronounced decrease in the rate of tetrahydrobiopterin dissocia tion (from 1.6+/-0.5 to 0.3+/-0.1 min(-1)). Low-temperature SDS gel el ectrophoresis showed that approx. 80% of the protein migrated as haemc ontaining dimer after preincubation with L-arginine and tetrahydrobiop terin. Gel-filtration chromatography yielded one peak with a Stokes ra dius of 6.8 +/- 0.04 nm, corresponding to a hydrodynamic volume of 1.3 2 x 10(-24) m(3), whereas haem-deficient preparations (approx. 0.3 equ ivalent per subunit) contained an additional protein species with a hy drodynamic radius of 5.1+/-0.2 nm and a corresponding volume of 0.55 x 10(-24) m(3), suggesting that haem availability regulates eNOS dimeri zation.