Biomedical subjects

P J Birckbichler

Publications and source records attributed to P J Birckbichler.

At least 19 recordsLinked to original sources

PubMed2000

Loss of tissue transglutaminase as a biomarker for prostate adenocarcinoma.

BACKGROUND: Additional molecular tissue biomarkers for prostate carcinoma are needed to stratify patients with clinically suspicious findings, such as an elevated prostate specific antigen (PSA) with a negative biopsy, according to risk. METHODS: Prostate tissues from 43 cancer cases and 47 controls with no evidence of cancer were labeled for transglutaminase by immunohistochemistry. Immunoreactivity was quantified using the Autocyte Pathology Workstation. In addition, quantitative fluorescence image analysis was used to compare transglutaminase concentrations in cells obtained by fine-needle aspiration from excised prostates. Loss of gene expression was evaluated by reverse transcriptase-polymerase chain reaction and growth with 5-azacytidine. RESULTS: Visually, benign glands from controls generally expressed tissue transglutaminase, whereas regions with adenocarcinoma generally were negative. With quantitative immunohistochemistry, 41 of 43 adenocarcinoma of the prostate (CaP) cases expressed lower mean percentage areas positive for transglutaminase than did 30 of 30 benign prostatic hyperplasia (BPH) and 17 of 17 prostatitis cases (P < 0.0001; odds ratio [OR], 1577; 95% confidence interval (CI), 74-33, 820; relative risk [RR], 25; 95% CI, 6-95). Quantitative immunofluorescence of 3277 cells collected by FNA from 19 CaP cases and 645 cells from 5 cases of BPH showed that the mean content of transglutaminase was 93 femtograms (fg) for the CaP-derived cells and 138 fg for the BPH cells (P < 0.0001). Receiver operating curve analysis of the immunohistochemistry data showed an optimized threshold produced 95% sensitivity with 100% specificity. Growth of LNCaP cells with 5-azacytidine failed to stimulate transglutaminase expression, suggesting that loss of expression was likely not attributable to promoter methylation. CONCLUSIONS: Measurements of transglutaminase on tissue sections provides additional diagnostic information that is potentially useful for risk assessment of patients with suspicious clinical findings, such as nodules or positive PSA and negative biopsies, without overdetecting disease.

Adenocarcinoma↗

PubMed1999

Synthesis, structure-activity relationships, and RARgamma-ligand interactions of nitrogen heteroarotinoids.

Three heteroarotinoids containing a nitrogen atom in the first ring and a C-O linking group between the two aryl rings were synthesized and evaluated for RAR and RXR retinoid receptor transactivation, tumor cell growth inhibition, and transglutaminase (TGase) induction. Ethyl 4-(N,4,4-trimethyl-1,2,3,4-tetrahydroquinolinyl)benzoate (1) contained an N-CH(3) group and activated all retinoid receptors except for RARgamma. Inceasing the hydrophobicity around the rings with analogues ethyl 4-(N,4,4,7-tetramethyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (2) [7-methyl group added] and ethyl 4-(4,4-dimethyl-N-isopropyl-1,2,3, 4-tetrahydroquinolin-6-oyloxy)benzoate (3) [NCH(CH(3))(2) group at C-4] increased the potency and specificity for RARalpha, RARbeta, and RXRalpha, compared to 1, but had little effect on RXRbeta and RXRgamma activation. Although 1 and 3 were unable to activate RARgamma, 2 did activate this receptor with efficacy and high potency equal to that of 9-cis-retinoic acid (9-c-RA). All three heteroarotinoids exhibited 5-8-fold greater specificities for RARbeta over RARalpha. In addition, esters 1-3 inhibited the growth of two cell lines each derived from cervix, vulvar, ovarian, and head/neck tumors with similar efficiencies to that of 9-c-RA through a mechanism independent of apoptosis. The vulvar cell lines were the most sensitive, and the ovarian lines were the least sensitive. Ester 2 was similar to 1 and 3 except that 2 was a much more potent growth inhibitor of the two vulvar cell lines, which is consistent with strong RARgamma activation by 2 (but not by 1 and 3) and the high levels of RARgamma expression in skin. All three heteroarotinoids induced production of TGase, a marker of retinoid activity in human erythroleukemic cells. Esters 2 and 3 were the more potent TGase activators than 1, in agreement with the stronger activation of the RAR receptors by 2 and 3. The biological activities of these agents, and the RARgamma potency of 2 in particular, demonstrate the promise of these compounds as pharmaceutics for cancer and skin disorders.

Antineoplastic Agents↗

PubMed1997

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity.

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.

Animals↗

PubMed1997

The role of transglutaminase in the rat subtotal nephrectomy model of renal fibrosis.

Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P </= 0.01. Levels of epsilon-(gamma-glutamyl) lysine cross-link correlated well with the renal fibrosis score throughout the 120 observation days (r = 0.78, P </= 0.01). Tissue homogenates showed no significant change in overall transglutaminase activity (14C putrescine incorporation assay) unless adjusted for the loss of viable tubule cells, when an increase from 5.77+/-0.35 to 13.93+/-4.21 U/mg DNA in cytosolic tissue transglutaminase activity was seen. This increase was supported by Western blot analysis, showing a parallel increase in renal tissue transglutaminase content. Immunohistochemistry demonstrated that this large increase in epsilon-(gamma-glutamyl) lysine cross-link and tissue transglutaminase took place predominantly in the cytoplasm of tubular cells, while immunofluorescence also showed low levels of the epsilon-(gamma-glutamyl) lysine cross-link in the extracellular renal interstitial space. The number of cells showing increases in tissue transglutaminase and its cross-link product, epsilon-(gamma-glutamyl) lysine appeared greater than those showing signs of typical apoptosis as determined by in situ end-labeling. This observed association between tissue transglutaminase, epsilon-(gamma-glutamyl) lysine cross-link, and renal tubulointerstitial scarring in rats submitted to SNx suggests that tissue transglutaminase may play an important role in the development of experimental renal fibrosis and the associated loss of tubule integrity.

Animals↗

PubMed1996

Alpha1-adrenergic receptor signaling via Gh is subtype specific and independent of its transglutaminase activity.

Tissue transglutaminase (TGase II) is a Ca2+- and thiol-dependent enzyme that catalyzes the post-translational modification of proteins via the formation of epsilon(gamma-glutamyl) lysine bonds. We have shown previously that the GTP-binding protein, Gh, is a TGase II that mediates intracellular signaling by the alpha1B-adrenergic receptor (AR) (Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Mison, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596). Here, we evaluated the ability of Gh as compared with Gq to mediate receptor-stimulated inositol phosphate turnover by the three alpha1-subtypes (alpha1A, alpha1B, and alpha1D). In addition, we questioned if the transglutaminase function of Gh is involved in its receptor signaling activity. A mutant form of a human TGase II cDNA in which the codon for the active site cysteine (Cys277) was replaced by serine was cloned into the mammalian expression vector pMT2'. Compared with wild-type TGase II, no transglutaminase activity was observed with transient transfection of this Cys-->Ser mutant in COS-1 cells. However, like wild-type TGase, the Cys-->Ser mutant mediated receptor-stimulated inositol phosphate turnover when cotransfected with an alpha1B-AR cDNA. Galphaq supported alpha1-AR-mediated inositol phosphate turnover by all three receptor subtypes. By contrast, although both the wild-type and Cys-->Ser construct mediated receptor signaling by the alpha1B AR and alpha1D AR, the alpha1A-AR was unable to interact with Gh. However, a Gh-dependent signaling phenotype could be rescued by a chimeric alpha1A construct in which the third intracellular loop of the alpha1A-AR was replaced by that of the alpha1B-AR. Thus, the signaling function of Gh is independent of its transglutaminase activity and is alpha1-AR subtype specific. This subtype specificity of the interaction between alpha1 ARs and Gh involves important determinants in their third intracellular loops.

Amino Acid Sequence↗

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↗

PubMed1996

Enhanced apoptosis in transformed human lung fibroblasts after exposure to sodium butyrate.

Simian virus-transformed human cells, WI-38 VA13A, showed a dose-dependent induction of apoptosis and reduction in cell numbers after exposure to sodium butyrate. Apoptosis was confirmed by ApopTag staining, isolation of apoptotic envelopes, and immunofluorescent staining with an antibody specific for apoptotic envelopes. Examination of the cell cultures by phase contrast and fluorescent microscopy revealed the presence of enlarged cells that displayed a more flattened morphology and morphological changes in the nucleus of cells exposed to sodium butyrate. Cell proliferation assays showed control and sodium butyrate cultures were synthesizing DNA and excluded any cytotoxic effects of sodium butyrate. Flow cytometry results indicated an increase in the number of aneuploid cells following sodium butyrate treatment. There was a decrease in the percentage of cells in G2/M in the diploid populations, but an increase in the percentage of cells in G2/ M in aneuploid populations. This human in vitro model system suggests a mode of action for the therapeutic effects of sodium butyrate, which have been observed in the topical treatment of neoplastic cells and reversal of symptom in ulcerative colitis, namely, the induction of apoptosis.

Aneuploidy↗

PubMed1995

Complex formation between gamma-immunoglobulin and calmodulin in calcium-free conditions.

We show that gamma-immunoglobulin (IgG) binds calmodulin (CaM) in a Ca(2+)-independent manner, with Kd value of (1.7 +/- 0.5) x 10(-7) M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca2+ greatly diminished the interaction between IgG and CaM, with IC50 = 8-9 microM. These data give a novel insight into protein-protein interactions.

Animals↗

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↗

PubMed1995

Purification and characterization of rat brain transglutaminase.

Transglutaminase (TGase) catalyzes an acyl-transfer reaction between peptidyl glutamine residues and primary amines including the epsilon-amino group of lysine residues in protein. TGase in the neuronal system has been suggested to be involved in neurotransmitter release, long-term potentiation, and so forth. In order to study the mammalian brain TGase at the molecular level, TGase was purified to apparent homogeneity from Sprague-Dawley rat brain, using DEAE ion exchange, and heparin and alpha-casein affinity column chromatographies. The brain TGase was concentrated 11,400-fold and had a specific activity of 11,000 nmol/h/mg protein. The purified protein migrated on SDS-PAGE to a position corresponding to a molecular size of approximately 75 kDa. The brain TGase activity was Ca(2+)-dependent (EC50 approximately 0.28 mM), and its Km values for putrescine and N,N-dimethylcasein were 0.26 and 0.065 mM, respectively. GTP inhibited the brain enzyme activity 100-fold more potently than ATP did, and the enzyme was photolabeled with [alpha-32P]8-azido-GTP, suggesting that the brain TGase is a member of the GTP-binding protein family. Monoiodo-acetate and cystamine potently inhibited the enzyme activity, suggesting that cysteine residue(s) are essential for brain TGase. Zn2+ inhibited the enzyme, while Mg2+ was not inhibitory. The rat brain TGase reacted only weakly with monoclonal and polyclonal antibodies against tissue-type TGase such as guinea pig liver TGase and human red blood cell TGase, while it did not react with antibodies against non-tissue types of TGase, namely human epidermal TGase and human coagulation factor XIIIa. The results suggest that rat brain TGase shares major characteristics with tissue-type TGase, but is nevertheless distinct from tissue-type TGase.

Affinity Labels↗

PubMed1993

Site-directed mutagenesis of human tissue transglutaminase: Cys-277 is essential for transglutaminase activity but not for GTPase activity.

Transglutaminases (EC 2.3.2.13) catalyze an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues in protein. Purified human erythrocyte transglutaminase was found to have another activity, i.e., GTP hydrolysis. Treatment of the enzyme with iodoacetamide, a cysteine-directed reagent, caused a 94% loss of TGase activity within 8 min, but no significant loss of GTPase activity. Cys-277, a known residue which is selectively modified by iodoacetamide, was replaced with Ser by site-directed mutagenesis to assess the role of the Cys-277 in the transglutaminase/GTPase activities. Wild-type cDNA, coding for human endothelial cell transglutaminase, and its C277S-mutated cDNA were cloned into a plasmid vector that contained a promoter from phage T7, and then expressed in Escherichia coli. The wild-type recombinant enzyme was indistinguishable from human erythrocyte transglutaminase in mobility on a SDS-polyacrylamide gel, immunoreactivity and catalytic activities for transglutaminase and GTPase. However, the recombinant enzyme was not blocked at the N-terminal alanine residue, as is the case in the naturally occurring erythrocyte enzyme. The C277S mutant enzyme showed no transglutaminase activity, but had Km and kcat values for GTPase activity that were comparable to those of wild-type recombinant and natural erythrocyte enzymes. These results demonstrate that Cys-277 is essential for transglutaminase activity, but not for GTPase activity, and that N-terminal blocking of tissue-type transglutaminase is not critical for either transglutaminase or GTPase activities.

Binding Sites↗

PubMed1992

A retinoic acid-inducible mRNA from human erythroleukemia cells encodes a novel tissue transglutaminase homologue.

A 1.9-kilobase (kb) cDNA for a new transglutaminase protein has been cloned and sequenced from retinoic acid-induced human erythroleukemia (HEL) cells. Full-length cDNA analysis reveals an open reading frame coding for a polypeptide of 548 amino acid residues with a molecular weight of 61,740. The deduced amino acid sequence exhibited 98% identity to the human cellular transglutaminase sequence. The cysteine at position 277 in the active site and the putative Ca(2+)-binding pocket at residues 446-453 of cellular transglutaminase are conserved. Such evidence predicts that the encoded protein product is likely to be a transglutaminase homologue (TGase-H). Immunoprecipitation of the in vitro translation products from a synthetic TGase-H mRNA and from total protein of cultured erythroleukemia HEL cells revealed a protein with a molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blot analysis of HEL cells and normal human fibroblast cells WI-38 using a cellular TGase probe detected the 1.9- and 4.0-kb RNA species at a relative abundance of 1:3 and 1:7, respectively. The 3'-end of the human cellular transglutaminase mRNA was also cloned and sequenced to allow comparison to the 3'-end of TGase-H reported here. This new piece gives a full length of 4012 nucleotides (4.0 kb) for human cellular transglutaminase. Comparison of the 5'-end (bases 1-1747) of the 1.9- and 4.0-kb cDNA sequences revealed a very high degree of identity. Beginning with base 1748, the sequences diverge showing no homology. The divergence point correlates with known intron-exon consensus boundaries indicative of alternative splicing.

Amino Acid Sequence↗

PubMed1992

Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.

2-Acetylaminofluorene↗

PubMed1992

Putative nucleotide binding sites of guinea pig liver transglutaminase.

Three peptides corresponding to glycine-rich internal sequences of the guinea pig liver transglutaminase molecule were synthesized. These were peptide 1 (amino acid residues 520-544), peptide 2 (amino acid residues 345-367) and peptide 3 (amino acid residues 45-69). All of the synthetic peptides demonstrated significant binding ability for both ATP and GTP. Peptide 1 was the best protector of transglutaminase activity from both ATP and GTP inhibition, while peptides 2 and 3 protected the activity only from GTP inhibition. The data shown here lead us to propose putative binding site(s) for ATP and GTP guinea pig liver transglutaminase.

Adenosine Triphosphate↗

PubMed1992

Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.

A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.

Amines↗

PubMed1991

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.

Amino Acid Sequence↗

PubMed1991

Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues.

Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links but includes the incorporation of polyamines into suitable protein substrates as well. The covalent isopeptide crosslink is stable and resistant to proteolysis, thereby increasing the resistance of tissue to chemical, enzymatic, and mechanical disruption. The plasma transglutaminase, factor XIIIa, is formed at sites of blood coagulation and impedes blood loss by stabilizing the fibrin clot. The squamous epithelium constituting the protective callus layer of skin is formed by the action of keratinocyte transglutaminase (TGK) and epidermal transglutaminase (TGE). The tissue transglutaminase (TGC) is a cytoplasmic enzyme present in many cells including those in the blood vessel wall. TGC function is unknown, although it could function to stabilize intra- and extra-cellular molecules in a wide variety of physiologic or pathologic processes. The amino acid sequences of factor XIII, TGC, and TGK establish them as a homologous gene family and also reveal a striking homology to the erythrocyte membrane protein, band 4.2. This review summarizes the current information on structures, functions, and evolution of the most prominent members of this gene family.

Amino Acid Sequence↗

PubMed1990

Significance of transglutaminase-catalyzed reactions in growth and development of filarial parasite, Brugia malayi.

A novel form of transglutaminase enzyme [EC 2.3.2. 13] was identified in adult worms of Brugia malayi. The molecular size of this enzyme was 22-kilodaltons as determined by Western blot and immunoprecipitation, using a monoclonal (CUB 7401) or polyclonal antibodies against guinea-pig liver tissue transglutaminase. The enzyme was present in female worms only; adult males contained no detectable levels of the enzyme peptide. Possible involvement of transglutaminase-catalyzed reactions in growth and survival of filarial parasites was studied by using various enzyme-specific pseudosubstrates. Presence of these inhibitors resulted into a significant inhibition of microfilariae production and release by gravid female worms in a dose-dependent manner. These results suggest that transglutaminase-catalyzed reactions are essential for development of in utero growing embryos to mature microfilariae.

Animals↗