Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Efforts to develop an improved assay for plasma and tissue transglutaminase have led us to a convenient, sensitive microtiter plate assay for coagulation factor XIII using human fibrinogen as an immobilized substrate. Factor XIII was activated in the presence of calcium, thrombin, and immobilized fibrinogen and then assayed by adding biotinylcadaverine. The reaction was terminated by adding EDTA and the level of incorporated biotin was measured with streptavidin-beta-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0.01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinogen-coated plates gave more than 100-fold increase in sensitivity compared with N,N-dimethylcasein-coated microtiter plates. The intraassay coefficient of variation was less than 5% (n = 12) and interassay less than 6% (n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin gamma-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII.
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PMID:A microtiter assay for factor XIII using fibrinogen and biotinylcadaverine as substrates. 769 7

One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-alpha, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-alpha increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-alpha, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-alpha. Gel shift experiments using nuclear extracts from TNF-alpha-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-kappa B motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-kappa B sequence showed an attenuated response to TNF-alpha treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a transgenic mouse line containing the mouse tTG promoter driving the beta-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-alpha. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-alpha when those cells overexpress a transdominant, negative mutant NF-kappa B subunit. Because TNF-alpha expression is upregulated in hepatic inflammation, the data suggest TNF-alpha-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.
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PMID:TNF-alpha modulates expression of the tissue transglutaminase gene in liver cells. 948 75

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.
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PMID:Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain. 1052 59

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.
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PMID:Apoptosis-linked in vivo regulation of the tissue transglutaminase gene promoter. 1117 60

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.
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PMID:Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor. 1287 89

Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.
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PMID:The promoter of the mouse tissue transglutaminase gene directs tissue-specific, retinoid-regulated and apoptosis-linked expression. 1455 66

We have previously described that tissue transglutaminase (tTG) is a high level phenotypic biomarker in prostate cancer, which is down regulated in prostate cancer and surrounding premalignant field compared to benign prostate glands. To understand the function of tTG in prostate cancer, we sought to identify proteins that interact with the transglutaminase moiety of tTG using a human prostate cancer complementary deoxyribonucleic acid library in a Yeast 2-Hybrid system. The Yeast 2-Hybrid experiments identified a strong and novel interaction between the transglutaminase moiety and protein kinase A anchor protein 13 (AKAP13), which was quantified by beta-galactosidase assay, confirmed in vitro by immunoprecipitation experiments using PC3 prostate cancer cell lysates, and in vivo colocalization was confirmed by immunofluorescence studies in PC3 cells. Because AKAP plays a major role in protein kinase A and Rho protein mediated signaling, functional studies are underway to elucidate the significance of tTG-AKAP13 interaction in prostate cancer.
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PMID:Tissue transglutaminase interacts with protein kinase A anchor protein 13 in prostate cancer. 1630 Nov 18