Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization

Homodimer formation is essential for the normal activity of endothelial NO synthase (eNOS). Structural uncoupling of eNOS with generation of enzyme monomers is thought to contribute to endothelial dysfunction in several vascular disorders including aging. However, published reports on healthy arteries using low-temperature SDS-PAGE has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Experiments therefore investigated optimal approaches for analyzing expression of eNOS monomers and dimers, using human cultured aortic endothelial cells and aortas from young and aged F344 rats. The results demonstrate that published differences in treatment of cellular lysates can significantly impact the relative expression of several eNOS species including denatured monomers, partially-folded monomers, dimers and higher-order ...