January 1, 1990Proceedings of the National Academy of Sciences3,308 citationsOpen Access
Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme.
View Full Paper
Key Points
- To isolate, purify, and biochemically characterize the enzyme responsible for synthesizing nitric oxide from arginine.
- Purified nitric oxide synthetase from rat cerebellum using 2',5'-ADP affinity chromatography eluted with NADPH.
- Assessed cofactor requirements including calmodulin, NADPH, and Ca2+ for enzymatic activity.
- Determined molecular weight and subunit structure via SDS/PAGE analysis.
- Nitric oxide synthetase was purified 6000-fold to homogeneity from rat cerebellar tissue.
- Enzymatic conversion of arginine to nitric oxide and citrulline was shown to strictly require calmodulin, NADPH, and Ca2+.
- The purified enzyme migrated as a single 150-kDa band on SDS/PAGE, indicating a native monomeric structure.
Abstract
Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neutrophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citrulline requires NADPH and Ca2+. We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affinity column eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer.