Particulate and Soluble Forms of the Inducible Nitric Oxide Synthase Are Distinguishable at the Amino Terminus in Raw 264.7 Macrophage Cells

Abstract We report here on the characterization of soluble and particulate forms of the inducible nitric oxide synthase in RAW 264.7 macrophage cells. Stimulation of these cells with E. coli lipopolysaccharide and interferon-gamma resulted in a significant induction of nitric oxide synthase activity with approximately 20% of the total activity localized to the cell membrane. Like the soluble enzyme form, the membrane-associated nitric oxide synthase activity was inhibited by N G -monomethyl-L-arginine and did not require the addition of calcium. Both protein forms were immunoreactive on Western blots with antibodies specifically recognizing the carboxyl terminus of the protein. In contrast, antibodies specific for the amino terminus recognized inducible nitric oxide synthase from the cytosol, but failed to recognize the membrane-associated protein. Thus, macrophage cells are capable of expressing two forms of the inducible nitric oxide synthase that are definable by an intracellular distribution that correlates to antigenic differences at the amino terminus.