Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.
- 1 December 1991
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 88 (23), 10480-10484
- https://doi.org/10.1073/pnas.88.23.10480
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 approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded 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.Keywords
This publication has 29 references indexed in Scilit:
- Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.Proceedings of the National Academy of Sciences of the United States of America, 1991
- A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblastsAmerican Journal of Physiology-Heart and Circulatory Physiology, 1991
- Purification of nitric oxide synthase from rat macrophagesJournal of Biological Chemistry, 1991
- Endothelial cells have a particulate enzyme system responsible for EDRF formation: Measurement by vascular relaxationBiochemical and Biophysical Research Communications, 1991
- Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophilsJournal of Biological Chemistry, 1991
- Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.Proceedings of the National Academy of Sciences of the United States of America, 1991
- Purification of a Ca2+/calmodulin‐dependent nitric oxide synthase from porcine cerebellumFEBS Letters, 1990
- Arginine is a physiological precursor of endothelium-derived nitric oxideEuropean Journal of Pharmacology, 1988
- ACTIVATION OF PURIFIED SOLUBLE GUANYLATE-CYCLASE BY ENDOTHELIUM-DERIVED RELAXING FACTOR FROM INTRAPULMONARY ARTERY AND VEIN - STIMULATION BY ACETYLCHOLINE, BRADYKININ AND ARACHIDONIC-ACID1986
- Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries.Circulation Research, 1986