Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

Hepatocytes are capable of repeated inducible NO synthase (NOS) expression, which occurs under inflammatory and stress conditions. This NOS expression regulates a number of cellular functions as well as cell viability. To better understand the posttranslational mechanisms that regulate the fate of NOS in these cells, we characterized the NOS distributed within peroxisomes. The selective permeabilization of membranes (plasma vs. peroxisomal) confirmed that there are cytosolic and peroxisomal pools of NOS in cytokine-stimulated hepatocytes and that the MOS protein associates with peroxisome. Detergent solubilization of the membrane fraction released NOS to the soluble fraction. NOS localized to membrane fraction is predominantly monomeric, but dimerization is partially reconstituted rapidly upon incubation with tetrahydrobiopterin. The reconstituted NOS exhibits a lower specific activity than NOS isolated from the soluble pool. Depletion of intracellular tetrahydrobiopterin with an inhibitor of de novo pterin synthesis resulted in a predominance of monomeric MOS without a greater relative distribution of MOS to the peroxisomal pool. Thus, NOS exists in a least two pools in hepatocytes: a soluble pool composed of both active dimer and monomer and a peroxisomal pool of monomeric MOS. MOS might localize to peroxisomes in long-lived cells such as hepatocytes as a protective mechanism to remove incompetent enzyme.