Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization

摘要: The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. protein soluble and highly active (0.2-0.7 micromol of l-citrulline. mg-1.min-1), contained 0. 77+/-0.01 equivalent haem per subunit, showed Soret maximum at 396 nm, exhibited only minor uncoupling NADPH oxidation in the absence l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values 147+/-24.1 nM 52+/-9.2 for specific tetrahydrobiopterin presence 0.1 mM respectively. positive co-operative effect due pronounced decrease rate dissociation (from 1.6+/-0.5 3+/-0.1 min-1). Low-temperature SDS gel electrophoresis that approx. 80% migrated as haem-containing dimer after preincubation with Gel-filtration chromatography yielded one peak Stokes radius 6.8+/-0.04 corresponding hydrodynamic volume 1. 32x10(-24) m3, whereas haem-deficient preparations (approx. 0.3 subunit) an additional species 5.1+/-0.2 nm 0.55x10(-24) suggesting availability regulates eNOS dimerization.