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. 1997 Apr 1;323(Pt 1):159–165. doi: 10.1042/bj3230159

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.

B M List 1, B Klösch 1, C Völker 1, A C Gorren 1, W C Sessa 1, E R Werner 1, W R Kukovetz 1, K Schmidt 1, B Mayer 1
PMCID: PMC1218289  PMID: 9173876

Abstract

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

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Selected References

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