Isoform specific effects of the autoinhibitory element and the C-terminus of nitr

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DESCRIPTION (provided by applicant): Nitric oxide synthases play multiple roles in such diverse physiological processes as control of vascular tone, signal transduction in the central nervous system, and immune response. Temporally and/or spatially inappropriate production of nitric oxide (NO) leads to several different pathologies. The endothelial isoform of NOS (eNOS) controls vascular constriction and dilation and has effects on platelet aggregation, with pathologies including hypertension and other vascular diseases. Autoimmune mechanisms trigger NO production by iNOS, leading to apoptosis of pancreatic beta cells in both insulin-dependent and non-insulin-dependent diabetes mellitus. NOS is a large modular enzyme with a heme containing oxygenase domain and a three domain reductase component. Primary control of eNOS and nNOS is exerted through regulation of electron flux from NADPH to the oxygenase active site. The most important of several inputs is calcium/calmodulin (Ca+2/CaM), but NO synthesis is also influenced by phosphorylation and protein-protein interactions. Ca+2/CaM control requires the participation of control elements located in the reductase region. The most important of these is the autoinhibitory insertion in the FMN binding domain, but the C terminal extension is also influential. The proposed work is organized around five hypotheses: 1. The autoinhibitory element of constitutively expressed NOS (cNOS) has isoform-specific effects in the absence of the C-terminal tail 2. The C-terminal tail has isoform-specific effects in the absence of the AI. 3. The C-terminal tail has isoform-specific effects in the presence of the AI 4. The autoinhibitory element and the C-terminal tail modulate each other's effects 5. Autoinhibitory element components have isoform-specific effect(s). The project will examine these hypotheses by creating novel chimeral genes in which control elements will be exchanged with cognates in distantly related NOS enzymes. Related chimera have been produced in a number of laboratories, establishing the feasibility of the approach. By using a larger section of the naturally occurring variability of the control sequences, we hope to greatly extend our knowledge of the control mechanism. Experiments suitable for undergraduate participation will be used to evaluate control of NO synthesis and electron transfer in mutants. More sophisticated kinetic analysis will allow us to obtain deeper insights than previous experiments with control element chimera.