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.
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.
List
B M
BM
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Klösch
B
B
Völker
C
C
Gorren
A C
AC
Sessa
W C
WC
Werner
E R
ER
Kukovetz
W R
WR
Schmidt
K
K
Mayer
B
B
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D004730:Endothelium, Vascular / Q000201:enzymology*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D019255:NADPH Oxidases / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic; D018411:Spodoptera
D000975:Antioxidants; D001708:Biopterins; D006418:Heme; D019001:Nitric Oxide Synthase; D019255:NADPH Oxidases; C003402:sapropterin