Consequences of terbium (III) binding on the conformation and enzymatic activity of guinea pig liver transglutaminase.
Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10-200 fold molar excess of calcium ions (Ki for Tb(III) = 60 microM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 microM to 10 microM) inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 microM to 100 microM) promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl2 only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl2 solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.
Achyuthan
K E
KE
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Mary
A
A
Bhaerman
R
R
Greenberg
C S
CS
D000818:Animals; D005340:Fibrinogen / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008674:Metals, Rare Earth / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000494:pharmacology*; D011503:Transglutaminases / Q000302:isolation & purification / Q000378:metabolism*
D008674:Metals, Rare Earth; D013725:Terbium; D005340:Fibrinogen; D011503:Transglutaminases