[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jul 2004 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) 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 +/- 3.2 mmHg; P < 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Sep 2002 · Drug Metabolism Reviews
[Show abstract][Hide abstract] ABSTRACT: PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science 274, 774-778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem. 271, 19358-19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein in Escherichia coli. Recombinant PIN is a folded dimeric, mostly alpha-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163-245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell 82, 743-752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer-monomer contact region.
No preview · Article · Dec 1998 · Archives of Biochemistry and Biophysics