Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Abstract

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B.The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.Nitric oxide (NO) is synthesized by NO synthases (NOSs) during conversion of L-arginine to L-citrulline (for reviews, see refs. 1 and 2). Biochemical characterization of NOSs has distinguished two general classes: (i) constitutive, dependent on exogenous Ca2+/calmodulin, and (ii) inducible, independent of exogenous Ca2+/calmodulin. Analyses of cDNA clones have identified three distinct NOS genes in mammals (3-6): neuronal, endothelial, and macrophage. The neuronal and endothelial NOSs are constitutive, and the macrophage NOS is inducible. The nomenclature for these different isoforms used here is historical, as it is clear now that one or more isoforms can be present in the same tissue (7).As a diffusible, free-radical gas, NO is a multifunctional messenger affecting many diverse aspects of mammalian physiology (for review, see ref. 8), such as regulation of vascular tone, macrophage-mediated cytotoxicity, and cell-cell interactions in the nervous system, including synaptogenesis and apoptosis during development of the rat central nervous system (9) and during neuronal cell differentiation (10). NO also appears to be involved with long-term potentiation in hippocampus and with long-term depression in cerebellum, two forms of synaptic plasticity that may underlie behavioral plasticity (11-15). Consistent with these cellular studies, inhibition of NOS activity may disrupt learning (refs. 16-19, but see refs. 20 and 21).Many of the above results are based on pharmacological studies using inhibitors of NOS or donors of NO. Interpretations of such studies usually are limited because the drugs interact with more than one target and cannot be delivered to specific sites. A molecular genetic approach can overcome these problems, however, by disrupting a specific gene, the product of which may be one of the drug's targets. Recently, such an approach has been attempted in mice via generation of a knockout mutation of the neuronal NOS (nNOS) (22).While nNOS mutants appeared fully viable and fertile, minor defects in stomach morphology and hippocampal long-term potentiation were detected (22, 23). Taken together, the above reports suggest roles for NO in developmental and behav...

Critical role of nitric oxide-cGMP cascade in the formation of cAMP-dependent long-term memory

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Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

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