Optimized Purification and Activity Analysis of Endothelial Nitric Oxide Synthase (eNOS, NOS3)
Disciplines
Biochemistry, Biophysics, and Structural Biology
Abstract (300 words maximum)
Endothelial nitric oxide synthase (NOS3, also known as eNOS) plays a crucial role in vascular health by synthesizing nitric oxide (NO), which regulates vascular tone, blood pressure, and endothelial function. Despite its significance, producing active NOS3 for biochemical studies remains challenging. We developed an optimized protocol to yield full-length NOS3 in a highly purified and active state, validated by SDS-PAGE and spectrophotometric analysis. NOS3 is as a substrate of p38 alpha, we tested the ability of wild p38 alpha and two binding site variants to phosphorylate NOS3. Differential phosphorylation patterns enable us to predict the areas on p38 alpha that are likely to be physiologically relevant to the intracellular binding of NOS3 and p38. The successful purification of active NOS3 opens new avenues for investigating its structure-function relationships, regulatory mechanisms, and potential therapeutic applications in cardiovascular diseases. This optimized protocol provides a reliable means of obtaining recombinant NOS3 suitable for detailed biochemical and functional studies. Purified protein will facilitate future studies on NOS3 regulation and its role in endothelial health and vascular homeostasis. Our future research aims to elucidate the relationship between NOS3 and Sirtuin 6 (SIRT6), an NAD+-dependent deacetylase, to understand how SIRT6 modulates NOS3 function and its implications for endothelial health.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Carol Chrestensen
Optimized Purification and Activity Analysis of Endothelial Nitric Oxide Synthase (eNOS, NOS3)
Endothelial nitric oxide synthase (NOS3, also known as eNOS) plays a crucial role in vascular health by synthesizing nitric oxide (NO), which regulates vascular tone, blood pressure, and endothelial function. Despite its significance, producing active NOS3 for biochemical studies remains challenging. We developed an optimized protocol to yield full-length NOS3 in a highly purified and active state, validated by SDS-PAGE and spectrophotometric analysis. NOS3 is as a substrate of p38 alpha, we tested the ability of wild p38 alpha and two binding site variants to phosphorylate NOS3. Differential phosphorylation patterns enable us to predict the areas on p38 alpha that are likely to be physiologically relevant to the intracellular binding of NOS3 and p38. The successful purification of active NOS3 opens new avenues for investigating its structure-function relationships, regulatory mechanisms, and potential therapeutic applications in cardiovascular diseases. This optimized protocol provides a reliable means of obtaining recombinant NOS3 suitable for detailed biochemical and functional studies. Purified protein will facilitate future studies on NOS3 regulation and its role in endothelial health and vascular homeostasis. Our future research aims to elucidate the relationship between NOS3 and Sirtuin 6 (SIRT6), an NAD+-dependent deacetylase, to understand how SIRT6 modulates NOS3 function and its implications for endothelial health.