Endothelial nitric-oxide synthase. Evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system.
A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.
Chen
P F
PF
Vascular Biology Research Center, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Tsai
A L
AL
Berka
V
V
Wu
K K
KK
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000201:enzymology*; D006639:Histidine; D006801:Humans; D007074:Immunoglobulin G; D007700:Kinetics; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016324:Sequence Tagged Sites; D013053:Spectrophotometry; D018411:Spodoptera; D014162:Transfection; D014357:Trypsin
D002147:Calmodulin; D017931:DNA Primers; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D006639:Histidine; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D002118:Calcium