INACTIVATION OF NITRIC-OXIDE SYNTHASES AND CELLULAR NITRIC-OXIDE FORMATION BY N-6-IMINOETHYL-L-LYSINE AND N-5-IMINOETHYL-L-ORNITHINE

The kinetics of inactivation of affinity-purified nitric oxide synthas e isoforms by N-6-iminoethyl-L-lysine (NIL) and N-5-iminoethyl-L-ornit hine (NIO) has been examined. Each of the agents produced a time and c oncentration dependent first order inactivation of the nitric oxide sy nthase isoforms that required exposure of the NO synthase to drug unde r conditions that supported catalysis, consistent with the proposal th at these agents act as alternate substrate, mechanism-based inactivato rs. As measured at 100 mu M arginine, NIL and NIO were equally efficie nt as inactivators of the cytokine-inducible nitric oxide synthase exh ibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM(-1) min(-1) respectively. By contrast, NIL and NIO were less e fficient as inactivators of the constitutive neuronal nitric oxide syn thase isoform exhibiting apparent second order inactivation rate const ants of 0.79 and 8.4 mM(-1) min-' respectively. As measured at 100 mu M extracellular arginine, NIL, and NIO produced a time and concentrati on dependent inactivation of the NO synthetic capability of cytokine-i nduced murine macrophage RAW 264.7 cells exhibiting apparent second or der inactivation rate constants of 3.1 and 1.8 mM(-1) min(-1). The ina ctivated RAW cell NO synthetic capability was restored to 30% of its p retreatment value over a 3-h period despite the presence of cyclohexim ide. (C) 1998 Elsevier Science B.V. All rights reserved.