Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.

Abstract

The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to -135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts , by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endotheliumdenuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2 , calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.Endothelial cells synthesize endothelium-derived relaxing factor (EDRF) from L-arginine (1, 2). The pharmacological and biochemical properties of EDRF are mimicked by nitric oxide (NO) (3) or NO-containing compounds (4). These agents activate soluble guanylyl cyclase (5, 6) thereby increasing cGMP and causing relaxation of vascular smooth muscle (7-9), inhibition of platelet aggregation (10), and other effects (9, 11). EDRF/NO synthase has been purified and characterized from brain (12-14), polymorphonuclear neutrophils (15), and endotoxin/cytokine-induced macrophages (16,17). The endothelial, brain, and neutrophil EDRF/NO synthases are constitutive enzymes whereas the macrophage activity is expressed only after induction with endotoxin and/or a cytokine. The constitutive enzyme from brain is soluble and Ca2+/calmodulin-regulated whereas the enzyme from neutrophils has been described as soluble and Ca2+-dependent, but not calmodulin-dependent. Analysis with denaturing gel electrophoresis revealed a single band corresponding to 155 kDa and 150 kDa for the brain (12) and neutrophil (15) In endothelial cells, a particulate Ca2+/calmodulin-regulated enzyme accounts for >95% of the total EDRF synthase activity (18,19). We now report the purification to homogeneity (denaturing molecular mass of 135 kDa) of this constitutive particulate EDRF synthase and characterize the enzyme as NADPH-and (6R)-5,6,7,8-tetrahydrobiopterin (BH4)-dependent. Some of these observations have been reported in abstract form (20,21).

Up‐regulation of endothelial nitric oxide synthase expression by cyclic guanosine 3′,5′‐monophosphate

“…The membrane bound ecNOS was solubilized as previously described [20,21] with slight modifications. Briefly, the bovine pulmonary endothelial cells that were exposed to 8-bromo-cyclic GMP or the vehicle for 48 h were scraped and washed in PBS and then suspended and homogenized in ice-cold 50 mM Tris.HCl (pH 7.4), containing 0.1 mM EDTA, 0.1 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 ktM leupeptin and 1/~M pepstatin A.…”

Superoxide as a Messenger of Endothelial Function

“…One of these isoenzymes, endothelial NO synthase (eNOS), is constitutively expressed in endothelial cells . This enzyme is largely membrane associated as a result of NH 2 -terminal myristoylation (Pollock et al, 1991), a reaction which regulates enzymatic biological activity (Sessa et al, 1993;Busconi and Mitchel, 1993). Endothelium-dependent relaxation 8-2 declines with increasing age (Tschudi et al, 1996).…”

Role of copper,zinc-superoxide dismutase in catalyzing nitrotyrosine formation in murine liver

“…Protein concentration was measured by the Lowry method [38]. As an indicator of NO production, plasma nitrate and nitrite concentrations were measured [39,40] to test if the impact of SOD1 knockout on hepatic protein nitration was related to NO formation. …”