Nucleotide binding by the erythrocyte transglutaminase/Gh protein, probed with fluorescent analogs of GTP and GDP.

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.