Abstract
Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.
Highlights
Guanosine 5“triphosphate (GTP) was foundto in- conformational change in the active site of the enzyme which hibitguineapig liver transglutaminase activity as stimulated its catalytic activity (Gross and Folk, 1973)
Effect of Guanine Nucleotide Derivatives on Purified Guinea Pig Liver Transglutaminase Activity-GTP, GTP-yS, GMP, cyclic GMP, GDP, andpolyguanylic acid were each tested at a concentrationof 1mM to determine theirrelative affects on guinea pig liver transglutaminase activity
In our examination of guanine nucleotides and their effects on guinea pig liver transglutaminase, the prototype for intracellular tissue transglutaminase, we found that GTP, GTP-y-S, and GDP significantly inhibited the activity of purified enzyme
Summary
Guanosine 5“triphosphate (GTP) was foundto in- conformational change in the active site of the enzyme which hibitguineapig liver transglutaminase activity as stimulated its catalytic activity (Gross and Folk, 1973). To CTP-agarose columns, and the biwnadsininghibited Recent studies have implicated nucleotide triphosphates as by the presence of calcium ions. GTP to transglutaminase was demonstrated bypho- activity. Loewy et al (1981a, 1981b) reported an increase in toaffinity labeling with 8-azidoguanosine ~ ’ - [ Y - ~ ’ Pt]he formation of intracellular (7-glutamy1)-e-lysylcross-links triphosphate, which was inhibited by the presence of when ATP was added to extracts from tissue-cultured embry-. GTPorCaClZ.GTP inhibited trypsin proteolysis of onic chick heart myofibrils or from cultures of the slime mold, guineapig liver transglutaminasewithout affecting Physarium polycephulum. Pro- ase is stabilized by the presence of ATP during purification teolytic protection was reversedbytheadditionof of the enzyme but not required for stability once purified calcium. This study demonstrates that GTP binds to (Brenner and Finn Wold, 1978). Cohen et al (1980) reported transglutaminase and function in concert to ture and function
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