NOS, isoforms

NOS (eNOS, NOS IH, NOS3). Classically, nNOS and eNOS were considered constitutive enzymes, whereas iNOS is cytokine-induced. Recent evidence suggests that nNOS and eNOS are also subject to important regulation of expression [1 ]. Within the human species, amino acid sequences of the three NOS isoforms share 52-58% identity. Each isoform is well conserved across mammalian species (>90% amino acid identity for nNOS and eNOS, >80% for iNOS). NOS enzymes exist in organisms as low as nematodes, protozoa, and even in plants (Fig. 1). 

All NOS isoforms utilize L-arginine as the substrate, and molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates. Flavin adenine dinucleotide (FMN), flavin mononucleotide (FAD), and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) are cofactors of the enzyme. All NOS isoforms contain heme and bind calmodulin. In nNOS and eNOS,... 

In contrast to well-described neuronal, endothelial, and inducible isoforms of NO synthase, mitochondrial NO synthase (mtNOS) is still not fully identified enzyme. As early as 1995, it has been reported that this NOS isoform is located to the inner mitochondrial membrane in... 

It is generally thought that analogs of the native substrate of NOS isoforms, L-arginine, bind to the active site as an inhibitor or a substrate. Many analogs of L-arginine have been synthesized, and the functions of these compounds have been analyzed. 

Fig. 10.7 Inhibitory potencies of N-alkyl-S-isothioureas towards three NOS isoforms. The inhibitory potencies are expressed as 1/Kj. N.T. means not tested.

Figure 1 provides a schematic representation of the three major NOS isoforms. The variation in size is due primarily to extensions and modifications at the N-terminal region where the greatest heterogeneity is observed. This variation is related to targeting of NOS to specific cellular... 

Fig. 1. The overall reaction catalyzed by NOS and a schematic representation of the three main NOS isoforms. L-Arg is first converted to JV-hydroxy-L-Arg in a P450-like monooxygenation reaction requiring two electrons and one O2 molecule. In the second step, Ai-hydroxy-L-Arg to NO and L-citrulline, only one electron and one O2 molecule are required. nNOS, neuronal NOS (neural system) iNOS, inducible NOS (immune system) eNOS, endothelial NOS (cardiovascular system).

A comparative analysis of the dimer interface between NOS isoforms is important since there are questions on significant variation in dimer stability between isoforms (84, 85). The dimer interface is extensive with approximately 2700 A of surface area buried per monomer. The interface contacts involve a mix of nonpolar and polar interactions, including hydrogen bonding. Approximately 60% of the interface in both iNOS and eNOS is hydrophobic, although the higher resolution eNOS... 

In general, the NOS isoforms can be grouped into two classes as determined by their expression in cells. Certain NOS isoforms, termed constitutive, appear to be expressed at a fairly constant level in their host cells. However, these isoforms are inactive in their native state and require that the Ca -binding protein calmodulin associate with them in order to generate NO (Schmidt et al., 1991 Busse and Mulsch, 1990 Bredt and Snyder, 1994). Thus NO production by constitutive NOS isoforms is often linked to Ca +-mediated signal transduction cascades that involve soluble guanylate cyclase, which is a hemeprotein that is activated by NO (Arnold et al., 1977). 

Other NOS isoforms, termed inducible, are typically not present in cells until the cell is exposed to immunostimulants of bacterial or host origin (Nathan,... 

NOS isoforms have now been purified from several sources, some of their properties are listed in Table 1. 

The rat brain and macrophage NOS isoforms are soluble, dimeric enzymes, each comprised of two identical subunits (Stuehr et al., 1991a Schmidt et al., 1991). Denatured molecular weights for the various NOS isoforms range from 130 to 150 kDa. The endothelium NOS is of unknown quaternary composition, and localizes primarily in the membrane fraction (Pollock et al., 1991), due to posttranslational myristoylation near the N-terminus (Lamas etaL, 1992). Phosphorylation consensus motifs are present on the brain and endothelial isoforms, with phosphorylation possibly down regulating NOS activity or preventing localization onto the membrane (Bredt et al., 1992 Michel et al., 1993). 

Although the reported specific activities for the NOS isoforms have varied widely, this may be due to differences in assay conditions (e.g., failure to add cofactors whose requirement was not known at the time) or differential stabilities of the preparations. When brain and macrophage isoforms are assayed in the... 

Overall sequences, similarity between the constitutive endothelium and neuronal NOS isoforms is approximately 50%, and both are roughly 50% identical to the inducible macrophage NOS (Xie et al., 1992 Lamas et al., 1992). Studies have located the genes for human neuronal, endothelial, and inducible NOS on... 

How the NOS isoforms compare to these related dual flavin enzymes is a matter of ongoing investigation. Characterization of the neuronal NOS revealed that it normally exists in its one-electron reduced form and maintains an air-stable, flavin semiquinone radical (Stuehr and Ikeda-Saito, 1992), as seen for NADPH-cytochrome P450 reductase. It is unknown which flavin in NOS contains the odd electron, although precedent argues that it probably resides on... 

Studies with the macrophage NOS were the first to show that NO synthesis was partially dependent on added Hbiopterin (Kwon et ai, 1989 Stuehr et al., 1990), which is a redox active cofactor utilized by the aromatic amino acid hydroxylases (Nichol et al., 1985). The requirement for H4biopterin has since been expanded to include all NOS isoforms studied to date. Mayer et al. (1990)... 

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