INACTIVATION OF MACROPHAGE NITRIC OXIDE SYNTHASE ACTIVITY BY NG-METHYL-L-ARGININE

Summary..N=O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N=O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of.N=O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by.N=O. NG-Methyl-L-arginine was a competitive inhibitor of.N=O synthase activity derived from activated murine macrophages with a Ki of 6.2 PM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 ’ C the kinact was 0.050 mint. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage.N=O synthase. 0 1991 ACxkrnlC mess, Inc. Many cells are now known to synthesize nitric oxide (.N=O) by the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine (1). Two types of.N=O synthases have been identified: a constitutively expressed Ca2+/calmodulin-dependent form isolated from ra