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1.
Two types of transglutaminase in the PC12 pheochromocytoma cell line. Stimulation by sodium butyrate 总被引:5,自引:0,他引:5
Transglutaminases are a class of enzymes capable of covalently cross-linking both intracellular and extracellular proteins. The activity of tissue transglutaminase is known to decrease precipitously following neoplastic transformation, and it has been hypothesized that transglutaminase may be involved in growth regulation. We have found that the differentiation promoter sodium butyrate is able to cause a marked increase in transglutaminase activity in PC12 pheochromocytoma cells in a time- and dose-dependent manner. This increased transglutaminase activity is associated with growth arrest, as well as with striking morphological changes including increased cell adhesion. The transglutaminase induced by sodium butyrate appears to be tissue transglutaminase, based on its cytosolic localization, thermal lability at basic pH, and elution profile on anion-exchange chromatography. Untreated PC12 cells contain only small amounts of transglutaminase which resembles epidermal transglutaminase, an enzyme previously described only in skin. In contrast to sodium butyrate, nerve growth factor did not stimulate tissue transglutaminase in PC12 cells, although it, too, caused growth arrest. It is hypothesized that transglutaminase may be involved in certain morphological changes accompanying cellular differentiation and neoplastic transformation, rather than in growth regulation per se. 相似文献
2.
Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes 总被引:3,自引:0,他引:3
M D George T M Vollberg E E Floyd J P Stein A M Jetten 《The Journal of biological chemistry》1990,265(19):11098-11104
This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis. 相似文献
3.
Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids 总被引:4,自引:0,他引:4
Rabbit tracheal epithelial cells undergo terminal cell division, start to express a squamous phenotype, and form cross-linked envelopes when reaching the plateau phase of the growth curve. This terminal differentiation is accompanied by a 20-30-fold increase in the activity of the cross-linking enzyme transglutaminase. This activity is found almost solely in the particulate fraction of homogenized cells and can be solubilized by nonionic detergents. This transglutaminase crossreacts with a monoclonal antibody raised against type I transglutaminase, but does not react with an antiserum against type II transglutaminase. The tracheal transglutaminase contains a protein subunit of approximately 92 kDa. The omission of epidermal growth factor from the medium or the addition of fetal bovine serum, conditions that induce terminal cell division and expression of a squamous phenotype, enhance transglutaminase activity. High calcium concentrations only stimulate transglutaminase activity after the cells become committed to terminal cell division. Retinoids, which inhibit the expression of the squamous phenotype but not terminal cell division, inhibit the enhancement in transglutaminase activity induced by either confluency or serum, indicating that this enzyme activity is under the control of retinoids. Some retinoids are active at concentrations as low as 10(-12) M. The ability of retinoids to inhibit transglutaminase activity correlates well with their capacity to bind to the retinoic acid-binding protein. Our results show that the increase in transglutaminase activity correlates with the induction of the terminal differentiated phenotype and suggest that this enzyme can function as a marker for this program of differentiation of rabbit tracheal epithelial cells in culture. Our results identify the transglutaminase as type I transglutaminase and are in agreement with the concept that this transglutaminase is involved in the formation of cross-linked envelopes. 相似文献
4.
Expression of tissue transglutaminase in the developing chicken limb is associated both with apoptosis and endochondral ossification 总被引:5,自引:0,他引:5
The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction. 相似文献
5.
The Ca2+-dependent tissue transglutaminase is widely distributed in various tissues and has been reported to participate in many cellular growth and differentiation processes. In the past decade, tissue transglutaminase is also identified as a G protein, G(alphah), for intercellular signaling. To further characterize testicular transglutaminase, the rat testicular transglutaminase was purified by ammonium sulfate precipitation, DEAE ion-exchange, heparin-agarose, and GTP-agarose affinity chromatographies. This purification protocol resulted in a 8400-fold enrichment of the enzyme with a reproducible 15% yield. The purified enzyme showed as a single band of 78kDa on SDS-polyacrylamide gel. Western blot analysis using anti-liver tissue transglutaminase monoclonal antibody also recognized the enzyme, indicating it is a t-TGase in nature. The Km values of purified testicular transglutaminase for putrescine and N,N-dimethylcasein were determined to be 35 and 17 microM, respectively. Its transglutaminase cross-linking activity was strongly inhibited by EGTA, GTP, polyamines, and cystamine, as well as moderately by ATP and NaCl. The enzyme exhibited a magnesium-dependent GTP-hydrolyzing capacity, but its GTP-binding activity did not require magnesium. Furthermore, the enzyme activity was found to be closely related with the first wave of spermatogenesis. Thus, testicular transglutaminase is speculated to participate in the event of spermatogenesis. In conclusion, the purified testicular transglutaminase displays property of either the tissue-type transglutaminase, or the GTP-binding and hydrolyzing characteristics. The activity of testicular transglutaminase is age-dependent, greatly stimulated during the first wave of spermatogenesis. 相似文献
6.
S H Yuspa T Ben H Hennings U Lichti 《Biochemical and biophysical research communications》1980,97(2):700-708
Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate. 相似文献
7.
Separation by anion exchange chromatography of detergent extracts from a poorly metastatic HSV-2-induced hamster fibrosarcoma, its highly metastatic variant and a highly metastatic rat fibrosarcoma indicated the presence of an inactive form of transglutaminase antigen, when eluent fractions were assayed for transglutaminase activity and antigen. This inactive antigenic transglutaminase was clearly separable from the particulate and cytosolic forms of the transglutaminase enzyme. Unlike tumours, its presence could not be demonstrated in extracts from normal rat liver. Measurement of activity levels during tumour growth indicated that the progression of the two highly metastatic tumours was accompanied by a decrease in cytosolic transglutaminase activity, whilst the activity of this enzyme form remained constant in the poorly metastatic tumour. Measurement of antigen levels indicated an inverse relationship between the level of inactive transglutaminase and the level of cytosolic transglutaminase activity, suggesting that the two forms are inter-related. Gel filtration indicated the molecular weight of the inactive form to be greater than both the particulate and cytosolic forms, and it was estimated to be 120,000. Partial proteolysis of the semi-purified inactive form, by either trypsin or thrombin, led to its activation and to the appearance of a transglutaminase similar in molecular weight and ionic mobility, both by anion-exchange chromatography and electrophoresis, to the cytosolic transglutaminase. 相似文献
8.
The polyglutamine-expanded N-terminal region of mutant huntingtin causes neurodegeneration in Huntington's disease (HD). Neuronal intranuclear and cytosolic inclusions composed of mutant huntingtin are found in brains of HD patients. Because tissue transglutaminase cross-links proteins into filamentous aggregates and polypeptide-bound glutamines are primary determining factors for tissue transglutaminase-catalyzed reactions, it has been hypothesized that tissue transglutaminase may contribute to the formation of these aggregates. In this report immunohistochemical and biochemical methods were used to demonstrate that tissue transglutaminase expression and transglutaminase activity are elevated in HD brains in a grade-dependent manner. In the striatum, tissue transglutaminase activity was significantly increased in the grade 3 HD cases compared with controls. When normalized to the neuronal marker calbindin D28k, immunoblot analysis revealed that in the striatum the levels of tissue transglutaminase were significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue transglutaminase immunoreactivity was markedly increased in all grades as compared with controls. In the superior frontal cortex, tissue transglutaminase activity was significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue transglutaminase levels were elevated in HD grades 2 and 3 cases. Tissue transglutaminase immunoreactivity within the superior frontal neocortex was also greater in all the HD cases compared with controls. These data clearly indicate that tissue transglutaminase is elevated in HD brain and may play a role in the disease process. 相似文献
9.
Mark A. Baumeister Michael A. Chattergoon 《Biotechnology & genetic engineering reviews》2013,29(1):205-244
Abstract Microbial transglutaminase is an important enzyme in food processing for improving protein properties by catalyzing the cross-linking of proteins. Recently, this enzyme has been shown to exhibit wider potential application in tissue engineering, textiles and leather processing, site-specific protein conjugation and wheat gluten allergy reduction. The production of microbial transglutaminase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering during the last three decades. More recently, studies on the biological mechanism of transglutaminase synthesis have further contributed towards the understanding of microbial transglutaminase production by Streptomyces. This will further facilitate improving the production of recombinant microbial transglutaminase. In this paper, we will review the progress in bioprocess engineering and genetic engineering in microbial transglutaminase production. We will highlight our understanding of the biological mechanisms of microbial transglutaminase synthesis, including biotechnological approaches used based on these biological mechanisms as a way of improving transglutaminase production.We address in addition the future research needs for microbial transglutaminase production. 相似文献
10.
GTP hydrolysis by guinea pig liver transglutaminase 总被引:4,自引:0,他引:4
K N Lee P J Birckbichler M K Patterson 《Biochemical and biophysical research communications》1989,162(3):1370-1375
Homogeneous guinea pig liver transglutaminase was purified from a commercially available enzyme preparation by affinity chromatography on GTP-agarose. The purified transglutaminase exhibited a single band of apparent Mr = 80,000 on sodium dodecyl sulfate polyacrylamide gel and Western blotting and had enzyme activity of both transglutaminase and GTPase. The guinea pig liver transglutaminase has an apparent Km value of 4.4 microM for GTPase activity. GTPase activity was inhibited by guanine nucleotides in order GTP-gamma-S greater than GDP, but not by GMP. These results demonstrate that purified guinea pig liver transglutaminase catalyzes GTP hydrolysis. 相似文献
11.
12.
Tissue transglutaminase differentially modulates apoptosis in a stimuli-dependent manner 总被引:3,自引:0,他引:3
Tissue transglutaminase is a unique member of the transglutaminase family as it not only catalyzes a transamidating reaction, but also binds and hydrolyzes GTP and ATP. Tissue transglutaminase has been reported to be pro-apoptotic, however, conclusive evidence is still lacking. To elucidate the role of tissue transglutaminase in the apoptotic process human neuroblastoma SH-SY5Y cells were stably transfected with vector only (SH/pcDNA), wild-type tissue transglutaminase (SH/tTG) and tissue transglutaminase that has no transamidating activity but retains its other functions (SH/C277S). In these studies three different apoptotic stimuli were used osmotic stress, staurosporine treatment and heat shock to delineate the role of tissue transglutaminase as a transamidating enzyme in the apoptotic process. In SH/tTG cells, osmotic stress and staurosporine treatments resulted in significantly greater caspase-3 activation and apoptotic nuclear changes then in SH/pcDNA or SH/C277S cells. This potentiation of apoptosis in SH/tTG cells was concomitant with a significant increase in the in situ transamidating activity of tissue transglutaminase. However, in the heat shock paradigm, which did not result in any increase in the transamidating activity in SH/tTG cells, there was a significant attenuation of caspase-3 activity, LDH release and apoptotic chromatin condensation in SH/tTG and SH/C277S cells compared with SH/pcDNA cells. These findings indicate for the first time that the effect of tissue transglutaminase on the apoptotic process is highly dependent on the type of the stimuli and how the transamidating activity of the enzyme is affected. Tissue transglutaminase facilitates apoptosis in response to stressors that result in an increase in the transamidating activity of the enzyme. However, when the stressors do not result in an increase in the transamidating activity of tissue transglutaminase, than tissue transglutaminase can ameliorate the apoptotic response through a mechanism that is independent of its transamidating function. Further, neither the phosphatidylinositol-3-kinase pathway nor the extracellular-regulated kinase pathway is downstream of the modulatory effects of wild-type tissue transglutaminase or C277S-tissue transglutaminase in the apoptotic cascade. 相似文献
13.
E K LeMosy H P Erickson W F Beyer J T Radek J M Jeong S N Murthy L Lorand 《The Journal of biological chemistry》1992,267(11):7880-7885
It has been reported previously (Turner, P.M., and Lorand, L. (1989) Biochemistry 28, 628-635) that human erythrocyte transglutaminase forms a noncovalent complex with human plasma fibronectin near its collagen-binding domain. In the present study, we show by nondenaturing electrophoresis that guinea pig liver transglutaminase, similarly to the erythrocyte enzyme, forms a complex with human fibronectin. Studies of anisotropic shifts of fluorescein-labeled liver and erythrocyte transglutaminases, upon addition of fibronectin, indicated that both transglutaminases bind to fibronectin with a stoichiometry of about 2:1. Polymerization of fibrinogen by human erythrocyte transglutaminase was inhibited after complex formation with fibronectin. Complexes of fibronectin with either erythrocyte or liver transglutaminase were isolated by glycerol gradient zone sedimentation and examined by rotary shadowing electron microscopy. The globular transglutaminase could be readily identified binding to the thin fibronectin strand. The binding site for transglutaminase was within 5-10 nm of the N terminus of fibronectin, consistent with its proximity to the collagen-binding domain. Under some experimental conditions, the complex of fibronectin with erythrocyte transglutaminase appeared as a ring-shaped structure in which two transglutaminase molecules had probably dimerized. The molecular weight of the erythrocyte transglutaminase was determined by sedimentation equilibrium to be 71,440 +/- 830. 相似文献
14.
Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells 总被引:9,自引:0,他引:9
P J Davies M P Murtaugh W T Moore G S Johnson D Lucas 《The Journal of biological chemistry》1985,260(8):5166-5174
Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells. 相似文献
15.
Michael Friedrich Carlos Villena-Heinsen Jianping He Dieter Mink Jörg Reichrath Karin Reitnauer Werner Schmidt 《The Histochemical journal》1999,31(1):13-18
The localization and expression of transglutaminase K has been investigated immunohistochemically in normal cervix tissue (n=15) and in cervix carcinomas (n=23). The distribution of the transglutaminase K was compared with the staining patterns of cytokeratin 10, Ki-67, p53, and oestrogen and progesterone receptors in these tumours. Weak to strong membrane-bound immunoreactivity for transglutaminase K was detected in almost all cervix carcinomas analyzed. The immunostaining was heterogeneous, with visual differences between individual tumour cells. 66.7% of normal cervix tissues revealed no immunoreactivity for the transglutaminase K. In normal cervix tissue, the immunoreactivity was confined to upper cervix layers, predominantly to the superficial and intermediate cell layers. The intensity of both the immunostaining and the number of transglutaminase K-positive cells were upregulated in cervix carcinomas as compared to normal cervix tissue. When the coexpressions of transglutaminase K with markers of proliferation and differentiation were analyzed, no statistically significant correlation was found. Our findings indicate that (1) transglutaminase K is upregulated at the protein level in cervix carcinomas as compared to normal cervix tissue; (2) upregulation of the transglutaminase K in cervix carcinoma is not exclusively induced by alterations of epithelial differentiation or proliferation, but by different, unknown mechanisms; and (3) upregulation of transglutaminase K in cervix carcinomas may play an important role for the regulation of tumour invasive properties by modulating cell–cell interactions. 相似文献
16.
Komandoor E. Achyuthan Ann Mary Robin Bhaermani Charles S. Greenberg 《Molecular and cellular biochemistry》1989,85(1):57-65
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 µM). 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 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM 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. 相似文献
17.
Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes 总被引:5,自引:1,他引:4
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《The Journal of cell biology》1993,120(6):1461-1470
Calcifying cartilages show a restricted expression of tissue transglutaminase. Immunostaining of newborn rat paw bones reveals expression only in the epiphyseal growth plate. Tissue transglutaminase appears first intracellularly in the proliferation/maturation zone and remains until calcification of the tissue in the lower hypertrophic zone. Externalization occurs before mineralization. Subsequently, the enzyme is present in the interterritorial matrix during provisional calcification and in the calcified cartilage cores of bone trabeculae. In trachea, mineralization occurring with maturation in the center of the cartilage is accompanied by expression of tissue transglutaminase at the border of the hydroxyapatite deposits. Transglutaminase activity also shows a restricted distribution in cartilage, similar to the one observed for tissue transglutaminase protein. Analysis of tissue homogenates showed that the enzyme is present in growth plate cartilage, but not in articular cartilage, and recognizes a limited set of substrate proteins. Osteonectin is coexpressed with tissue transglutaminase both in the growth plate and in calcifying tracheal cartilage and is a specific substrate for tissue transglutaminase in vitro. Tissue transglutaminase expression in skeletal tissues is strictly regulated, correlates with chondrocyte differentiation, precedes cartilage calcification, and could lead to cross-linking of the mineralizing matrix. 相似文献
18.
Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin 总被引:15,自引:0,他引:15
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading. 相似文献
19.
Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase 总被引:8,自引:0,他引:8
Elevated transglutaminase activity and formation of cornified envelopes are markers of terminal differentiation in mouse epidermal cells. Epidermal transglutaminase catalyzes cornified envelope formation and in cultured cells is inducible by calcium ion or phorbol ester tumor promoters. Retinoic acid also induces transglutaminase activity but inhibits cross-linked envelope formation. This apparent paradox might be resolved by the observation that the retinoic acid-induced transglutaminase appears to be either a different enzyme or a markedly altered form of the epidermal enzyme. The retinoic acid-induced transglutaminase is soluble in aqueous buffers, is thermolabile at pH 9.0, 37 degrees C, and elutes from an anion exchange column at 0.4 M NaCl. In contrast, the epidermal enzyme is particulate and requires detergent for solubilization, is relatively thermostable, and elutes from the anion exchanger at 0.25 M NaCl. The retinoic acid-induced enzyme is probably identical with the "tissue" transglutaminase present in liver and in other cells. It is proposed that the transglutaminase induced by retinoic acid may play a role in the inhibition by retinoids of calcium and tumor promoter-induced differentiation. 相似文献
20.
Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity. 总被引:5,自引:0,他引:5
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Bovine aortic endothelial cells contain Ca2+-dependent tissue-type transglutaminase. Its activity in these cells was high, with apparent Km and Vmax. values with respect to putrescine of 0.203 mM and 18.5 nmol/min per mg of protein, and its activity was inhibited by the three competitive inhibitors dansylcadaverine, spermine and methylamine. The molecular mass of endothelial cell transglutaminase estimated by gel filtration chromatography was 88 kDa and it was immunoprecipitated by rabbit monospecific antiserum raised against rat liver transglutaminase. Its enzymic activity rose when the cell cultures reached confluence, and was further increased when their proliferation was arrested (synchronized at G0/G1 phase). Most of the enzymic activity was found in the 15,000 g soluble fraction, with only 4-22% of the activity found in the particulate fraction, depending on the state of cell proliferation. Examination of these cellular fractions by SDS/polyacrylamide-gel electrophoresis and immunoblotting revealed that at confluence endothelial cells have accumulated transglutaminase antigen in their 15,000 g particulate fraction. A series of experiments demonstrated the existence of a latent transglutaminase form in non-proliferating cells, and suggested that this might involve the formation of an inhibitory complex. Treatment of cell lysates and the 15,000 g particulate fraction with high salt concentration showed a significant increase in transglutaminase activity. Mixing experiments using the 100,000 g particulate fraction or purified rat liver transglutaminase on one hand and the cytosolic fraction on the other showed dose-dependent inhibition of the transglutaminase activity of the latter. It is concluded that endothelial cells contain a particulate fraction-residing inhibitor of transglutaminase which interacts via ionic interaction with the enzyme. 相似文献