Original articleα2-Plasmin Inhibitor is a Substrate for Tissue Transglutaminase: An in Vitro Study
Section snippets
Materials
Human thrombin (3000 U/mg), guinea pig liver tissue transglutaminase (1.8 U/mg), monodansylcadaverine, iodoacetamide, dithiothreitol, and glycine ethyl ester were obtained from Sigma Chemicals (St. Louis, MO, USA). Electrophoretic grade chemicals for SDS PAGE were from Bio-Rad (Richmond, CA, USA). [14C]glycine ethyl ester hydrochloride (2.2 GBq/mmol) was purchased from New England Nuclear (Boston, MA, USA). [3H]putrescine dihydrochloride (999 GBq/mmol), [14C] methylated marker proteins,
Incorporation of small Mr primary amines into α2PI by tTG
The covalent binding of different fluorescent or radio-labeled amine substrates (dansylcadaverine, [14C]glycine ethyl ester or [3H]putrescine) to α2PI by tTG is demonstrated on Fig. 1A (lanes b). The presence of a TG active-site inhibitor, iodoacetamide, completely prevented the incorporation of amine substrates into the protein (lanes a). The incorporation of labeled amines into α2PI was blocked by EDTA, a Ca2+ chelating agent (not shown on the gels), as well. The appearance of a faint higher M
Discussion
α2PI, a member of the serine-protease-inhibitor super-family (serpins), is the primary inhibitor of plasmin. It contains three regions of importance. From the N-terminus they are as follows: the region containing the cross-linking site; the region containing the reactive center; and the region with the plasminogen binding site at the C-terminal extension of the molecule. The covalent attachment of α2PI by FXIIIa to fibrin is essential in the protection of newly formed fibrin strands against the
Acknowledgements
This work was supported by grants from the Hungarian National Science Fund (OTKA 030406 and OTKA F020752), from the Hungarian Ministry of Health (ETT 321/96) and from the Hungarian Academy of Sciences.
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2006, Journal of AutoimmunityCitation Excerpt :NH3 liberated during the transglutaminase reaction in liquid phase is measured by a coupled glutamate-dehydrogenase reaction and consequent decrease of β-nicotinamide adenine dinucleotide phosphate (NADPH) [26]. The total volume of reaction mixture was 200 μl containing 10 mM dithiotreitol (DTT), 30 mM ethyl-amine, 0.75 mM adenosine 5′-diphosphate, 7.5 mM α-ketoglutarate, 0.8 mM NADPH, 22.5 U/ml glutamate dehydrogenase (EC 1.4.1.4, Roche, Mannheim, Germany), 5 mM CaCl2, 5 mM glutamine substrate peptide (representing a sequence from α2-plasmin inhibitor; provided by L Kárpáti, Dept. of Clinical Biochemistry and Molecular Pathology, University of Debrecen, Debrecen, Hungary), 2 μg GST-fused TG2 and 150 μg/ml or ten times of normalised concentrations of IgA or IgG in 120 mM HEPES buffer, pH 7.5 [27]. After 10 min preincubation at room temperature (enzyme and antibodies in the buffer) the reaction was performed at 37 °C for 30 or 40 min.
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2005, Thrombosis ResearchCitation Excerpt :PI serves as an acyl donor glutamine substrate in the reaction [31]. Tissue transglutaminase, which is another member of the same family and present in a number of cell types, also catalyzes the cross-linking reaction of PI to fibrin(ogen) through exactly the same mechanism as FXIIIa [44,45]; the cross-linking occurs between Gln2 of Asn-PI and Lys303 of the Aα-chain of fibrinogen. Tissue transglutaminase in vascular endothelial cells, which is thrombin-independent, may be released upon cell injury and cross-link PI to fibrinogen deposits in the vessel wall [46], thus possibly contributing to the development of atherosclerosis.