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Biomedical subjects

K E Achyuthan

Publications and source records attributed to K E Achyuthan.

At least 19 recordsLinked to original sources
PubMed2001

Rapid diagnostics: the detection of neuraminidase activity as a technology for high-specificity targets.

The accurate detection of influenza by clinical symptoms is challenging since multiple pathogenic viruses and bacteria mimic similar symptoms in a patient. With new and more effective influenza therapeutics available, there is a growing need for highly accurate and rapid diagnosis of influenza, particularly when the window of opportunity for proper treatment is measured in hours. A parallel technology, which is also used in the treatment of influenza, was developed for the rapid diagnosis of influenza by exploiting the enzymatic activity of influenza neuraminidase. This technology, which is called Pathozyme, offers the high specificity inherent from the conservation of the neuraminidase active site. The ZstatFlu test uses a small molecule derivative of sialic acid chemically coupled to a reporter group together with simple point-of-care reagents for directly detecting influenza from a patient specimen with high specificity. A second-generation platform technology using this neuraminidase detection system coupled with a more sensitive chemiluminescent reporter has been developed and formatted for reading on high-speed instant film. This modification resulted in a platform technology many-fold more sensitive than the former while maintaining its inherent high specificity. Preliminary data from a prototype tested during the mild 2000-2001 influenza season demonstrated that an optimized chemiluminescent test system could approach the accuracy of 14 day viral culture in a convenient 10-20 min test. This platform technology is currently being explored for the rapid detection of other pathogenic organisms where sensitivity, specificity and speed are essential in a point-of-care setting.

Amino Acid Sequence↗
PubMed2001

Comparative enzymology, biochemistry and pathophysiology of human exo-alpha-sialidases (neuraminidases).

This review summarizes the current research on human exo-alpha-sialidase (sialidase, neuraminidase). Where appropriate, the properties of viral, bacterial, and human sialidases have been compared. Sialic acids are implicated in diverse physiological processes. Sialidases, as enzymes acting upon sialic acids, assume importance as well. Sialidases hydrolyze the terminal, non-reducing, sialic acid linkage in glycoproteins, glycolipids, gangliosides, polysaccharides, and synthetic molecules. Therefore, a variety of assays are available to measure sialidase activity. Human sialidase is present in several organs and cells. Its cellular distribution could be cytosolic, lysosomal, or in the membrane. Human sialidase occurs in a high molecular-mass complex with several other proteins, including cathepsin A and beta-galactosidase. Multi-protein complexation is important for the in vivo integrity and catalytic activity of the sialidase. However, multi-protein complexation, the occurrence of isoenzymes, diverse subcellular localization, thermal instability, and membrane association have all contributed to difficulties in purifying and characterizing human sialidases. Human sialidase isoenzymes have recently been cloned and sequenced. Even though crystal structures for the human sialidases are not available, the highly conserved regions of the sialidase from various organisms have facilitated molecular modeling of the human enzyme and raise interesting evolutionary questions. While the molecular mechanisms vary, genetic defects leading to human sialidase deficiency are closely associated with at least two well-known human diseases, namely sialidosis and galactosialidosis. No therapy is currently available for either disease. A thorough investigation of human sialidases is therefore crucial to human health.

Amino Acid Sequence↗
PubMed1999

Synthesis of bromoindolyl 4,7-di-O-methyl-Neu5Ac: specificity toward influenza A and B viruses.

N-Acetylneuraminic acid (Neu5Ac) was converted into the methyl ester methyl ketoside-8,9-epoxy derivative (8). Methylation of 8 followed by deprotection gave 4,7-di-O-methyl-Neu5Ac (10). Compound 10 was converted into the corresponding methyl ester-chloroacetate derivative, which was subsequently coupled to 5-bromo-indol-3-ol to give the chromogenic product (13). Deprotection of 13 gave 5-bromo-indol-3-yl 4,7-di-O-methyl-Neu5Ac (5). The product 5 was specifically cleaved by sialidase from either influenza A or influenza B virus to give an indigo-blue precipitate, but was not cleaved by several bacterial or viral sialidases tested. The properties of product 5 relative to a fluorescent substrate for sialidase were also documented.

Arthrobacter↗
PubMed1998

Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain.

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.

Animals↗
PubMed1996

Hierarchies in the binding of human factor XIII, factor XIIIa, and endothelial cell transglutaminase to human plasma fibrinogen, fibrin, and fibronectin.

The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIIa), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIIa bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn approximately 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3-15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12-25-fold more than Fn. FXIIIa bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10-20-fold less than Fn. The Kds' for FXIIIa binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The Kd for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn] > [FXIIIa-Fgn] = [FXIIIa-Fbn] > [FXIII-Fbn] > [FXIII-Fgn] = [FXIIIa-Fn] > [Tg-Fbn] = [Tg-Fgn] > [FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.

Endothelium↗
PubMed1995

Immunochemical analyses of human plasma fibronectin-cytosolic transglutaminase interactions.

Fibronectin is a glycoprotein involved in cell adhesion, tissue organization and wound healing. Transglutaminase binding and covalent cross-linking of fibronectin are physiologically important reactions. We describe microtiter plate-based immunochemical methods to analyze cytosolic transglutaminase-human plasma fibronectin interactions. The method was sensitive, specific, species-independent and capable of simultaneously analyzing 96 samples for binding. Binding was time-, temperature- and concentration-dependent and demonstrable with either protein immobilized to the plastic. The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked immunosorbent assays. CaCl2 (8 mM) enhanced transglutaminase binding by two-fold. Molar concentrations of NaCl or millimolar concentrations of chloride salts of barium, copper or zinc inhibited binding by 50-60%. The binding was also competitively blocked by soluble fibronectin (IC50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 microM). Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a concentration-dependent inhibition of transglutaminase-fibronectin interactions (IC50 = 1.5 mM and 20 mM, respectively). A complex of [anti-transglutaminase IgG-transglutaminase-fibronectin-anti- fibronectin IgG] suggested that the binding sites and antibody epitopes could overlap, but are distinct and surface-exposed in the two proteins. Liver transglutaminase bound fibronectin 30-50% less compared to erythrocyte transglutaminase. Fibronectin-transglutaminase affinity was adequate for quantitating either antigen in lysates of lung fibroblasts, breast carcinomas or Escherichia coli. These immunochemical analyses will be useful for determining the affinity and mapping the domains involved in antibody recognition or protein-protein interactions using recombinant molecules of transglutaminase and fibronectin.

Animals↗
PubMed1994

Carboxyl-terminal truncation of recombinant factor XIII A-chains. Characterization of minimum structural requirement for transglutaminase activity.

A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity. delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate. There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462. The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants. The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant. The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold. delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound. In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.

Base Sequence↗
PubMed1994

A microtiter plate assay for factor XIII A-chain-fibrin interactions.

Factor XIII A-chain-fibrin interactions regulate factor XIIIa formation and fibrin cross-linking. A microtiter plate assay was developed for studying these interactions. Microtiter plate wells were coated with fibrinogen and converted to fibrin by thrombin. After blocking the wells with bovine serum albumin, factor XIII A-chain was added and binding was monitored by incubating first with anti-factor XIII followed by anti-rabbit IgG-alkaline phosphatase. Enzymatic hydrolysis of p-nitrophenyl phosphate was quantitated by the absorbance at 405 nm. BInding was specific, sensitive, rapid, saturable, and reversible, requiring only nanograms of either factor XIII or fibrin. Binding was time- and concentration-dependent and independent of divalent cations. The bound material was identified as factor XIII A-chain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Factor XIII binding was inhibited > 75% by 250 mM sodium chloride or 250 nM anti-factor XIII IgG. The method was also suitable for demonstrating binding using 0.8% plasma or with r-factor XIII expressed in Saccharomyces cerevisiae or Escherichia coli. This method is suitable for identifying the binding sites that are important for plasma factor XIII activation and factor XIIIa activity.

Animals↗
PubMed1994

Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E. coli.

The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains. We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector. A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E. coli. Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 or Met475. We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains. JM105 harboring pKF13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium. The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 micrograms of FXIII A-chains per liter of culture. The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast. These results document that FXIII A-chains can be readily expressed and purified from E. coli culture and that they retained properties similar to those of purified human factor XIII A-chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Factor XIIIa-derived peptides inhibit transglutaminase activity. Localization of substrate recognition sites.

Factor XIIIa is a transglutaminase that catalyzes intermolecular gamma-glutamyl-epsilon-lysyl bonds between fibrin and other proteins involved in hemostasis. We synthesized 25 peptides from various regions of factor XIIIa and studied their effects on cross-linking fibrin, N,N'-dimethylcasein, or fibronectin. We found that two peptides, Asn72-Asp97 (peptide-4) and Asp190-Phe230 (peptide-7), inhibited factor XIIIa cross-linking of these substrates. The other peptides did not inhibit factor XIIIa activity. The inhibition of cross-linking was reversed by excess substrate, indicating that the peptides were interacting with fibrin and not factor XIIIa. The peptides were not pseudosubstrates since they were not cross-linked to fibrin. The peptides did not modify the primary amine binding site as increasing the primary amine concentration did not reverse inhibition. Peptides-4 and -7 also had no effect on exposure of the active site of factor XIIIa and no synergistic inhibitory effects were detected. Peptides-4 and -7 had no effect on factor XIIIa binding to fibrin suggesting that the binding sites and the substrate recognition sites were distinct. Synthetic peptides containing shorter amino acid sequences of peptide-4 were inactive. In contrast, the amino-terminal (Asp190-Lys199, Tyr194-Tyr204) and the carboxyl-terminal (Lys221-Phe230) portions of peptide-7 were 20-60-fold less inhibitory compared to intact peptide-7. Peptides-4 and -7 also inhibited guinea pig liver tissue transglutaminase from cross-linking fibrinogen, N,N'-dimethylcasein, and fibronectin. In conclusion, we have identified two regions outside the active site pocket which are important for substrate recognition in factor XIIIa and tissue transglutaminase.

Amines↗
PubMed1992

A microtiter plate transglutaminase assay utilizing 5-(biotinamido)pentylamine as substrate.

Transglutaminases belong to an important family of enzymes involved in hemostasis, skin formation, and wound healing. We describe a technique for the measurement of transglutaminase activity using polystyrene microtiter plates coated with N,N'-dimethylcasein. The substrate 5-(biotinamido)pentylamine is covalently incorporated into N,N'-dimethylcasein by transglutaminase in a calcium-dependent reaction. The biotinylated product is detected by streptavidin-alkaline phosphatase and quantitated by measuring the absorbance at 405 nm following the addition of p-nitrophenyl phosphate. The assay is sensitive, specific, and linear at plasma factor XIIIa concentrations between 0.08 and 1.25 micrograms/ml and at purified guinea pig liver transglutaminase concentrations between 0.05 and 0.8 microgram/ml. The intra-assay coefficient of variation is less than 8%. The solid-phase assay was used to quantitate the transglutaminase activity in Escherichia coli extracts expressing recombinant factor XIII A-chains and to analyze factor XIIIa inhibitors. This method will facilitate the analysis of structure-function relationships of the transglutaminases using recombinant DNA methods. Furthermore, screening of natural and synthetic factor XIIIa inhibitors will be expedited by this solid-phase microtiter plate assay.

Amines↗
PubMed1989

Human mononuclear phagocyte transglutaminase activity cross-links fibrin.

The physiologic function of the monocyte transglutaminases is not known. In this study, we detected Factor XIII A-subunit antigen and "tissue" transglutaminase antigen in human monocytes by polyacrylamide gel electrophoresis and immunoblotting techniques. Flow cytometric analysis demonstrated that 27% and 49% of the total Factor XIII antigen in monocytes and human peritoneal macrophages, respectively, are expressed on the surface of the cells. Monocytes maintained in culture for 8 days had a 4-fold increase in Factor XIIIa activity and a 3.2-fold increase in the amount of Factor XIII antigen/mg cell protein. However, there was no increase in the "tissue" transglutaminase activity or antigen levels in cultured monocytes. In addition, we identified a Factor XIII deficient individual who does not express Factor XIII activity or antigen in plasma, platelets, monocytes, lymphocytes or erythrocytes. Intact monocytes from normal donors were able to cross-link fibrin formed in the plasma from the Factor XIII deficient individual. This suggests that transglutaminase activity expressed by peripheral blood monocytes may play a physiologic role in cross-linking fibrin during blood clotting or inflammation.

Antigens, Surface↗
PubMed1989

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.

Animals↗
PubMed1989

Tb(III)-ion-binding-induced conformational changes in platelet factor XIII.

Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.

Animals↗
PubMed1988

Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen).

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.

Amino Acid Sequence↗
PubMed1988

Vitronectin is a substrate for transglutaminases.

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.

Cross-Linking Reagents↗
PubMed1988

The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin.

The present study represents detailed investigations into the nature of interactions between an intracellular "tissue" transglutaminase and a plasma protein, fibrinogen. We demonstrate a specific, saturable, and reversible binding of transglutaminase to fibrin(ogen). The binding was time- and temperature-dependent, was independent of divalent metal ions, did not require the release of either fibrinopeptide A or B, and was partially inhibited by the presence of sodium chloride or plasma proteins, properties similar to Factor XIII binding to fibrin(ogen). Both Factor XIII and liver transglutaminase also shared similar binding sites on fibrinogen, the A alpha- and the B beta-chains. The binding characteristics of liver transglutaminase were thus similar to Factor XIII binding to fibrin, but there were also important differences. Scatchard analyses of the binding data indicated that the affinity of liver transglutaminase (Kd = 4.17 x 10(-7) M) was at least 40-fold weaker compared with the affinity of Factor XIII to fibrinogen. Consequently, a 20-fold molar excess of Factor XIII a-chains specifically and completely inhibited the binding of liver transglutaminase to des-A-fibrinogen. The association between liver transglutaminase and fibrin(ogen) was also critically controlled by the conformational states of the two proteins. Substances capable of altering the conformation of either transglutaminase (such as guanosine 5'-triphosphate) or of fibrinogen (such as the tetrapeptide Gly-Pro-Arg-Pro and Fragment D) disrupted binding. Excess CaCl2 was able to counteract the effects of guanosine 5'-triphosphate on transglutaminase binding to fibrin. In contrast, Factor XIII binding to fibrin was unaffected by either guanosine 5'-triphosphate, CaCl2, or Gly-Pro-Arg-Pro, suggesting a more stable association between the two proteins. The physiologic implications of transglutaminase-fibrin(ogen) interactions are discussed.

Animals↗