Dependent Transglutaminase Research Articles

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1–10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Characterization of Endogenous Transglutaminase Enzyme of Yellow Pike Conger’s Liver

Transglutaminases have been found in various living organism, such as mammals, plants, <br />microorganisms, and marine organisms including fishes. Transglutaminases have many various functions<br />such as food properties, non-food properties and pharmacologies. This research aimed to characterize<br />transglutaminase that obtained from byproducts of yellow pike conger (Congresox talabon) such as<br />catadromous fish of yellow pike conger’s liver. The characteristic of transglutaminase had the possibility<br />to know the optimum condition in application of transglutaminase from liver of yellow pike conger such<br />as food processing industry, non-food industry and pharmacological application. A yield of yellow pike<br />conger’s liver was 0.88±0.11%. Specific activity of remang fish liver weighing 10 grams was 1,375 U/mg.<br />Characteristic crude transglutaminase from liver of yellow pike conger had optimum pH at 7.5; optimum<br />temperature at 50°C, transglutaminase activity increased on Mg<br />2+<br /> ion dependent and crude transglutaminase <br />had molecular weight of 27.48; 37.00; 69.51; 78.89; 88.18; 108.45; 134.10; dan 172.12 kDa.<br /><br />

Dermatan sulfate is a player in the transglutaminase 2 interaction network.

Transglutaminase 2 (TG2) is a multifunctional protein that is primarily engaged in cell adhesion/signaling or shows Ca2+-dependent transglutaminase activity in the extracellular space of tissues. This latter action leads to the cross-linking of the extracellular matrix (ECM) proteins. The enhanced extracellular expression of TG2 is associated with processes such as wound healing, fibrosis or vascular remodeling that are also characterized by a high deposition of dermatan sulfate (DS) proteoglycans in the ECM. However, it is unknown whether DS may bind to TG2 or affect its function. Using the plasmon surface resonance method, we showed that DS chains, especially those of biglycan, are good binding partners for TG2. The interaction has some requirements as to the DS structure. The competitive effect of heparin on DS binding to TG2 suggests that both glycosaminoglycans occupy the same binding site(s) on the protein molecule. An occurrence of the DS-TG2 interaction was confirmed by the co-immunoprecipitation of this protein with native decorin that is a DS-bearing proteoglycan rather than with the decorin core protein. Moreover, in vivo DS is responsible for both TG2 binding and the regulation of the location of this protein in the ECM as can be suggested from an increased extraction of TG2 from the human fascia only when an enzymatic degradation of the tissue DS was conducted in the presence of the anti-collagen type I antiserum. In addition, DS with a low affinity for TG2 exerted an inhibitory effect on the protein transamidating activity most probably via the control of the accessibility of a substrate. Our data show that DS can affect several aspects of TG2 biology in both physiological and pathological conditions.

Abstract B52: Calcium-sensitized tissue transglutaminase translocates to the plasma membranes and inhibits neuroblastoma cell migration

Abstract Tissue-transglutaminase (TG2) is a dual function G-protein with Ca2+-dependent transglutaminase activity and GTP-binding/hydrolyzing activity. TG2 is physiologically involved in cell differentiation, cell death, migration, invasion of cancer cells. In the extracellular matrix (ECM), TG2 mediates cell- ECM interactions through fibronectin and integrins and promotes cell attachment, migration and invasion. When present on the cytoplasmic side of the plasma membrane TG2 activates phospholipase C and RhoA-ROCK-2 signaling pathways that promote cell differentiation. While mechanism of TG2-induced cell differentiation is well characterized, the mechanism underlying TG2-mediated cell migration is not known. In present study, we explored the mechanism of how TG2 may be involved in cell migration by using wild type neuroblastoma cells (SH-SY5Y) which do not express TG2, neuroblastoma cells expressing exogenous TG2 (SHYTG2), and a transamidation-deficient TG2 containing SH-SY5Y cells (SHYmutant). Our cell migration assays (scratch assay and collagen-coated transwell assay) and cell invasion assay (matrigel-coated trasnwell assay) indicate that TG2 expressing cells exhibit higher migrating and invasive properties. To further investigate the role of TG2 in cell migration, we utilized a natural compound, resveratrol. Resveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic and phytoalexin compound present in grapes and blueberries and has shown promising results in the treatment of neuroblastoma. In our studies, resveratrol (1 μM -10 μM) significantly prevented migration and invasion of TG2-expressing cells. In migrating cells, resveratrol increases the immunoreactivity of TG2 without affecting the total TG2 protein level. In these cells, resveratrol increases calcium levels, and depletion of intracellular calcium by a calcium chelator, BAPTA, attenuate resveratrol-enhanced TG2 immunoreactivity. To further analyze resveratrol-induced cellular distribution and conformational states of TG2, we performed native gel electrophoresis and detected an additional TG2 protein band with slower migration in resveratrol-treated migrating cells. This TG2 form is non-phosphorylated, exclusively present in plasma membrane fractions and sensitive to intracellular Ca2+ concentrations suggesting a calcium requirement in TG2-regulated cell migration. The observed less mobility of transamidation-defective SH-SY5Y cells (SHYmutant) and the reduced migration of SHYTG2 cells in the presence of a TG2 transamidation inhibitor, monodensylcadevarine, in scratch assays further support the requirement of TG2 in cell migration. Our mechanistic studies suggest that resveratrol induces conformational changes in TG2 (compact to open structure form), and Ca2+-mediated TG2 association to plasma membrane is responsible for the inhibitory effects of resveratrol on cell migration. Together, present study indicates that TG2 plays a key role in neuroblastoma cell migration. Although higher concentrations of resveratrol (∼100 μM) induces cell death in cancer cells, the current study suggest that lower physiologically relevant dose of resveratrol (∼1 μM) prevent the migration and invasion of cancer cells from the primary site. Since retinoic acid treatment increases TG2 protein level and resveratrol inhibits the migration of TG2-expressing cells, this study also proposes the use of resveratrol with retinoic acid for the treatment of neuroblatoma. Citation Format: Ambrish Kumar, Jianjun Hu, Holly A. LaVoie, Kenneth B. Walsh, Donald J. DiPette, Ugra S. Singh. Calcium-sensitized tissue transglutaminase translocates to the plasma membranes and inhibits neuroblastoma cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B52.

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca(2+)-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.

Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis

A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca 2+, suggesting that the clotting reaction is catalyzed by a Ca 2+-dependent transglutaminase present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is the first report of a CP characterization in an Atlantic penaeid species. Further studies, including a molecular cloning approach would enable to detect which tissues express the gene of the clotting protein. It would be also useful to understand the mechanism by which the coagulation time is delayed in shrimps under stress conditions.

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage

Tissue transglutaminase (tTG) catalyzes a Ca 2+-dependent transglutaminase (TGase) activity which cross-links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg-GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM, but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.

Increase of the ornithine decarboxylase/polyamine system and transglutaminase upregulation in the spinal cord of aged rats

We have investigated changes in ornithine decarboxylase (ODC) activity and in polyamine levels in the central nervous system of aged rats. We measured a significant increase of ODC catalytic activity in the spinal cord from 30 month-old rats (+105%) as compared to 4 month-old rats. No changes were noticed in the cerebellum, cortex and hippocampus from the same animals. A related putrescine increase was measured in the spinal cord of 30 month-old rats (+168%), together with a smaller increase of spermidine (+33%). A parallel increase (+78%) of the Ca 2+-dependent transglutaminase activity was detected in the spinal cord of 30 month-old rats, while no changes were apparent in the cortex and cerebellum. Our observations indicate a possible role of the ODC/polyamine system during the normal process of ageing in rats and point to the spinal cord as the most sensitive area for this kind of modification. A possible role of protein polyamination by tranglutaminase is discussed.

Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

Triggered release of calcium from lipid vesicles: a bioinspired strategy for rapid gelation of polysaccharide and protein hydrogels

The bioinspired strategy of triggered release of Ca 2+ from liposomal compartments was used to induce rapid gelation of polysaccharide and protein-based hydrogels. Thermally triggerable liposomes were designed by entrapping CaCl 2 within liposomes constructed of 90% dipalmitoylphosphatidylcholine and 10% dimyristoylphosphatidylcholine. These liposomes released greater than 90% of entrapped Ca 2+ when heated to 37°C. A precursor fluid containing liposomes suspended in aqueous sodium alginate remained fluid for several days at room temperature but gelled rapidly when heated to 37°C, as a result of Ca 2+ release and formation of crosslinked Ca-alginate. Alternatively, thermally triggered Ca 2+ release from liposomes was used to activate enzyme-catalyzed crosslinking of proteins to form hydrogels. A mixture of Ca-loaded liposomes, fibrinogen, and a Ca 2+-dependent transglutaminase enzyme (either human recombinant FXIII or guinea pig liver transglutaminase) remained fluid indefinitely when stored at room temperature, but gelled rapidly when heated to 37°C. SDS-PAGE of the reaction mixture revealed that gelation was due to enzymatic crosslinking of the α and γ chains of fibrinogen, and oscillating rheometry revealed gel formation within 10 min of heating to 37°C. This new approach may be useful for developing rapidly gelling injectable biomaterials that can be stored at room temperature and injected in a minimally invasive manner into a body tissue or cavity, upon which rapid solidification would occur. This versatile bioinspired strategy could be utilized for the delivery of biomaterials for tissue repair and reconstruction, and local site-directed drug delivery.

Cytochrome P450–generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 μg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca2+]. This activated Ca2+-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca2+-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca2+]. This activates Ca2+-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity. (Hepatology 2000;32:303-311.)

Novel Protein-disulfide Isomerases from the Early-diverging Protist Giardia lamblia

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13) during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km) of the enzyme for the substrates N'N' dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5 mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

Properties of Purified Lens Transglutaminase and Regulation of its Transamidase/Crosslinking Activity by GTP

On account of its protein crosslinking activity, the Ca2+-dependent transglutaminase of the lens is likely to be involved in the formation of cataracts. We have now purified the rabbit lens enzyme to near homogeneity as judged by SDS-PAGE (Mr∼78 kDa), and a key feature of the procedure was the use of a highly selective affinity chromatographic step with a fibronectin fragment as ligand. The catalytic activity of the lens transglutaminase, measured by the incorporation of dansylcadaverine into dimethylcasein, was compared with those of two similar enzymes isolated from human red cells and from guinea pig liver, respectively. All three enzymes were inhibited by GTP, but the lens enzyme was most sensitive to inhibition by the nucleotide. Moreover, GTP was also shown to inhibit the formation of the ∼55 kDa βcrystallin dimers in the Ca2+-treated rabbit lens homogenate, proving that the nucleotide is a negative regulator for the crosslinking activity of transglutaminase in this tissue.

Detection of Ca2+-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants

Protein extracted from root and leaf tissue of the dicotyledonous plants pea (Pisum sativum) and broad bean (Vicia faba) and the monocotyledonous plants wheat (Triticum aestivum) and barley (Hordeum vulgare) were shown to catalyze the incorporation of biotin-labeled cadaverine into microtiter-plate-bound N', N'-dimethylcasein and the cross-linking of biotin-labeled casein to microtiter-plate-bound casein in a Ca2+-dependent manner. The cross-linking of biotinylated casein and the incorporation of biotin-labeled cadaverine into N',N'-dimethylcasein were time-dependent reactions with a pH optimum of 7.9. Transglutaminase activity was shown to increase over a 2-week growth period in both the roots and leaves of pea. The product of transglutaminase's protein-cross-linking activity, epsilon-(gamma-glutamyl)-lysine isodipeptide, was detected in root and shoot protein from pea, broad bean, wheat, and barley by cation-exchange chromatography. The presence of the isodipeptide was confirmed by reversed-phase chromatography. Hydrolysis of the isodipeptide after cation-exchange chromatography confirmed the presence of glutamate and lysine.

A Novel Calcium-Independent Enzyme Capable of Incorporating Putrescine into Proteins

A Ca++-independent enzyme capable of incorporating [3H]-putrescine into proteins was detected in the rat intestine mucosa. The Ca++-independent incorporation of [3H]-putrescine into proteins was temperature-, pH-, time-, and dose-dependent. However, this enzyme was absent in the gastric mucosa. Similar to testicular Ca++-dependent transglutaminase, the optimal pH of intestinal Ca++-independent enzyme was 9.0. At 10−5M or less putrescine concentrations, the Ca++-independent enzyme in an intestinal cytosol preparation showed a greater activity than did the Ca++-dependent transglutaminase. However, at higher putrescine concentrations, the latter showed a greater activity than did the former. Both the intestinal Ca++-dependent and independent enzymes were inhibited by cystamine, thermal labile at 50°C and precipitated by 30 to 50% saturation of ammonium sulfate. The fact that these two enzymes shared many similar characteristics, with the exceptions of Ca++-requirement, suggests that they may have similar active site and intrinsic molecular function(s).

Regulation of Human Tissue Transglutaminase Function by Magnesium-Nucleotide Complexes: IDENTIFICATION OF DISTINCT BINDING SITES FOR Mg-GTP AND Mg-ATP

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

Role of transglutaminase in keratinization of vaginal epithelial cells in oestrous cycling rats.

Aspects of the regulation of calcium dependent transglutaminase (TGase) enzyme in the terminally differentiating vaginal epithelial cells (VEC) from cycling and oestradiol primed ovariectomized (OVX) rats are described. There is a significant increase in the TGase activity and a quantitative rise in its cross-linked sigma(tau-glutamyl)lysine covalent product in the VEC of oestrus rats. A similar phenomena was also evident in the oestradiol primed OVX rats. However, in the VEC of the diestrus/unprimed OVX rats, the enzyme activity and its cross-linked product tend to be at its basal level. An increase of TGase activity in the keratinized layers of the oestrus VEC was observed. Immunohistochemical localization data parallel the biochemical measurements. These findings suggest that TGase activity may be associated with the differential status of the cell and primarily aids in the conferring structural stability and integrity to the stratified VEC. Aggregates of the keratin tonofilament bundles which are covalently cross-linked by the formation of sigma(tau-glutamyl)lysine in the oestrus vagina renders them resistant to denaturant and proteolytic treatments.

Diterpenoids from germander, an herbal medicine, induce apoptosis in isolated rat hepatocytes

BACKGROUND & AIMS: Germander was withdrawn from the market after its use for weight control caused an epidemic of hepatitis. Its toxicity was shown to be caused by diterpenoids and their cytochrome P4503A- mediated metabolic activation into electrophilic metabolites that deplete cellular thiols. The aim of the present study was to determine the mechanisms of cell death.METHODS: Isolated rat hepatocytes were incubated for 2 hours with germander diterpenoids (100 micrograms/mL).RESULTS: Diterpenoids decreased cell glutathione, increased cytosolic [Ca2+], activated Ca(2+)-dependent tissue transglutaminase forming a cross-linked protein scaffold, and caused internucleosomal DNA fragmentation and the ultrastructural features of apoptosis. Cell death was prevented by decreasing metabolic activation (with troleandomycin), preventing depletion of glutathione (with cystine), blocking activation of Ca(2+)-modulated enzymes (with calmidazolium), or inhibiting internucleosomal DNA fragmentation (with aurintricarboxylic acid). Apoptosis was increased and diterpenoids caused overexpression of p53 and interleukin 1 beta-converting enzyme in rats treated with dexamethasone (cytochrome P4503A inducer). Apoptosis was also increased by a diet deficient in sulfur amino acids.CONCLUSIONS: The germander furano diterpenoids cause apoptosis within 2 hours in isolated rat hepatocytes. Electrophilic metabolites may stimulate apoptosis by decreasing thiols, increasing [Ca2+], and activating Ca(2+)-dependent transglutaminase and endonucleases.(Gastroenterology 1997 Oct;113(4):1334-46)