Inactivation of nitric oxide synthases and cellular nitric oxide formation by N6-iminoethyl- l-lysine and N5-iminoethyl- l-ornithine

Abstract

The kinetics of inactivation of affinity-purified nitric oxide synthase isoforms by N 6-iminoethyl- l-lysine (NIL) and N 5-iminoethyl- l-ornithine (NIO) has been examined. Each of the agents produced a time and concentration dependent first order inactivation of the nitric oxide synthase isoforms that required exposure of the NO synthase to drug under conditions that supported catalysis, consistent with the proposal that these agents act as alternate substrate, mechanism-based inactivators. As measured at 100 μM arginine, NIL and NIO were equally efficient as inactivators of the cytokine-inducible nitric oxide synthase exhibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM −1 min −1 respectively. By contrast, NIL and NIO were less efficient as inactivators of the constitutive neuronal nitric oxide synthase isoform exhibiting apparent second order inactivation rate constants of 0.79 and 8.4 mM −1 min −1 respectively. As measured at 100 μM extracellular arginine, NIL and NIO produced a time and concentration dependent inactivation of the NO synthetic capability of cytokine-induced murine macrophage RAW 264.7 cells exhibiting apparent second order inactivation rate constants of 3.1 and 1.8 mM −1 min −1. The inactivated RAW cell NO synthetic capability was restored to 30% of its pretreatment value over a 3-h period despite the presence of cycloheximide.