Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.
is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 Ϯ 3.2 mmHg; P Ͻ 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.spontaneously hypertensive rat; cytochrome P-450; sodium 10-undecynyl sulfate; 20-hydroxyeicosatetraenoic acid CYTOCHROMES P-450 (CYPs) are major catalysts of renal arachidonic acid metabolism, and CYP-derived eicosanoids have been implicated in the regulation of vascular tone and renal function (9, 41). The major products of CYP-catalyzed arachidonic acid metabolism are the -hydroxylated metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) and four regio-and stereoisomeric epoxyeicosatrienoic acids (EETs). EETs are further hydrated to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) (53). The major CYP eicosanoid formed in rat and human renal microsomes is 20-HETE (8, 28). 20-HETE depolarizes vascular smooth muscle cells by inhibiting Ca 2ϩ -activated K ϩ channels and increases the conductance of L-type Ca 2ϩ channels, both effects leading to Ca 2ϩ entry and potent vasoconstriction (14,56). Additional roles for 20-HETE include mediation of the myogenic response of small cerebral and renal arteries to elevations in transmural pressure, and the autoregulation of cerebral and renal blood flow, glomerular filtration rate, and tubular glomerular feedback (13,22,58). In renal tubules 20-HETE inhibits Na ϩ -K ϩ -ATPase, a Na ϩ -K ϩ -2Cl Ϫ cotransporter, and 70-pS K ϩ channels, leading to natriuresis and diuresis (3,30,38). EETs and DHETs are generally considered vasodilatory, although they exhibit both vasodilatory and vasoconstrictive properties in vitro, depending on the vascular bed and species that are studied (20,55,57). A number of studies suggest that EETs are endothelium-derived hyperpolarizing factors that mediate vasodilation by activation of Ca 2ϩ -dependent K ϩ channels in vascular smooth muscle cells (6,7,10). Both EETs and DHETs can also mediate natriuresis and diuresis by inhibition of Na ϩ -K ϩ -ATPase in rat proximal tubules (38).The effects of CYP eicosanoids in the regulation of vascular tone and renal function have pr...
Cytochrome P450 and nitric oxide synthase (NOS) oxidize nitrogen atoms, although the substrates and transformations are highly restricted for NOS. The first reaction catalyzed by NOS is mediated by a P450-like ferryl species, although it is generated by a distinct process in which a tetrahydrobiopterin molecule in NOS serves as a transient electron donor. The second NOS reaction appears to be mediated by an iron dioxygen precursor of the ferryl species. The transient tetrahydrobiopterin radical formed in these reactions is quenched by electron transfer from the NOS flavin domain. Electron transfer from the flavins is controlled by the binding of calmodulin, the presence of peptide inserts in the flavin domain, the substrate structure, and phosphorylation of the enzyme.
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