Measurement of the Nitric Oxide Synthase Activity Using the Citrulline Assay

The free-radical gas nitric oxide (NO) recently has been identified as an important biological messenger molecule in both the central and peripheral nervous system. NO is generated by the enzyme NO synthase (NOS) by the oxidation of the amino acid L: -arginine. As a dissolved gas, NO is an unusual neurotransmitter. It is not packaged in synaptic vesicles and released by exocytosis upon membrane depolarization, but rather diffuses from its site of production to surrounding neurons where it acts directly on specific intracellular targets. The activity of NO terminates when it chemically reacts with a target substrate. Although all of the targets of NO are not yet known, NO can bind to the iron associated with heme groups or result in nitrosylation of proteins, leading to conformational changes. One of the best-described targets of NO in the central nervous system is the heme-containing protein guanylyl cyclase. NO is a relatively long-lived free radical and does not react readily with most cellular components. This allows it to diffuse to several surrounding neurons and integrate neuronal activity on a local scale. NO is involved in a number of physiological processes including morphogenesis and synaptic plasticity. However, under conditions in which NOS is overstimulated, excessive formation of NO may mediate cell injury in a variety of disorders of the nervous system that result in neurodegeneration (1).