title: Nucleotide binding by the erythrocyte transglutaminase/Gh protein, probed with fluorescent analogs of GTP and GDP.
abstract: GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A characteristic feature was the appearance of a major energy transfer band (lambda(exc, max) = 290 nm, lambda(em) = 444 nm) from protein tryptophans to the bound nucleotides. Quenching of the bound fluorophore (lambda(exc) = 360 nm, lambda(em) = 444 nm) by acrylamide was barely different from that of free ligand. However, major changes were observed in anisotropy, which was used to demonstrate a facile exchange between bound and free nucleotides and to evaluate affinity constants for the binding of methylanthraniloyl GTP and GDP to TG.
authors.affiliation: Department of Cell and Molecular Biology, and Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, IL 60611-3008, USA.
authors.forename: S N
authors.initials: SN
authors.lastname: Murthy
authors.forename: L
authors.initials: L
authors.lastname: Lorand
mesh_terms: D003432:Cross-Linking Reagents; D004912:Erythrocytes / Q000201:enzymology*; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006801:Humans; D013050:Spectrometry, Fluorescence; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D011503:Transglutaminases; D019204:GTP-Binding Proteins