Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity.
Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.
Department of Immunology, Cleveland Clinic, Ohio 44195, USA.
A
A
Presta
J
J
Liu
W C
WC
Sessa
D J
DJ
Stuehr
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D006801:Humans; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D013379:Substrate Specificity
D002147:Calmodulin; D009569:Nitric Oxide; D009249:NADP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III