TGase Activities Research Articles

Property and Function of a Novel Chitinase Containing Dual Catalytic Domains Capable of Converting Chitin Into N-Acetyl-D-Glucosamine.

A novel multifunctional chitinase (CmChi3)-encoding gene was cloned from Chitinolyticbacter meiyuanensis and actively expressed in Escherichia coli. Sequence analysis showed that CmChi3 contains two glycoside hydrolase family 18 (GH18) catalytic domains and exhibited low identity with well-characterized chitinases. The optimum pH and temperature of purified recombinant CmChi3 were 6.0 and 50°C, respectively. CmChi3 exhibited strict substrate specificity of 4.1 U/mg toward colloidal chitin (CC) and hydrolyzed it to yield N-acetyl-D-glucosamine (GlcNAc) as the sole end product. An analysis of the hydrolysis products toward N-acetyl chitooligosaccharides (N-acetyl COSs) and CC substrates revealed that CmChi3 exhibits endochitinase, N-acetyl-β-d-glucosaminidase (NAGase), and transglycosylase (TGase) activities. Further studies revealed that the N-terminal catalytic domain of CmChi3 exhibited endo-acting and NAGase activities, while the C-terminal catalytic domain showed exo-acting and TGase activities. The hydrolytic properties and favorable environmental adaptations indicate that CmChi3 holds potential for commercial GlcNAc production from chitin.

Changes of Ammonia-Metabolizing Enzyme Activity and Gene Expression of Two Strains in Shrimp Litopenaeus vannamei Under Ammonia Stress

Ammonia stress can inhibit the survival and growth, and even cause mortality of shrimp. In this study, ammonia-metabolizing enzyme activities and gene expression were compared between two strains of L. vannamei under different ammonia-N () concentrations (3.4, 13.8, and 24.6 mg/L). The results showed that elevated ammonia concentrations mainly increased glutamine synthetase (GSase) activities while inhibiting transglutaminase (TGase) activities in the muscle of both strains. Thus, we concluded that L. vannamei could accelerate the synthesis of glutamine from glutamate and to alleviate ammonia stress. Compared with the muscle, the hepatopancreas plays a major role in ammonia stress and might be a target tissue to respond to the ammonia stress. Compared to the control group, the treatment of high ammonia concentrations reduced the hepatopancreas TGase (TG) gene expression and increased the gene expression rates of glutamate dehydrogenase-β (GDH-β) and GSase (GS) in both the muscle and the hepatopancreas of the two strains (p < 0.05). These genes (GDH-β and GS) in strain B were not only expressed earlier but also at levels higher than the expression range of strain A. At the gene level, strain B showed a more rapid and positive response than strain A. These data might help reveal the physiological responses mechanisms of shrimp adapt to ammonia stress and speed up the selective breeding process in L. vannamei.

New Continuous Fluorometric Assay for Bacterial Transglycosylase Using Förster Resonance Energy Transfer

The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25–45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca 2+ and Mg 2+ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L −1, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.

Polyamines and transglutaminase actions: Polyamine und Transglutaminase-Wirkungen

Polyamines (putrescine, spermidine and spermine), secreted by the prostate gland, occur in high concentrations in the seminal fluid of many species, including man and rat. The physiological significance of seminal polyamines is still obscure, however. It has been postulated that polyamines may serve as amine donor substrates for transglutaminases (TGases), enzymes catalyzing protein crosslinking by the formation of gamma-glutamyl-lysine or bis(gamma-glutamyl)polyamine cross-bridges. We have analyzed TGase-activities and polyamine content of the various rat prostate lobes and rat seminal vesicles. Highest TGase activities were observed in the coagulating gland (anterior prostate) and the dorsolateral prostate, whereas very little TGase activity was present in the ventral prostate gland. In contrast, polyamine concentrations were highest in the ventral prostate but low in coagulating glands. Seminal vesicles, and in particular seminal vesicle secretions, contained low polyamine levels and intermediate TGase activity. Levels of protein-bound polyamines, not extractable by perchlorid acid, did not correlate with TGase-activities in ventral prostate and coagulating glands, suggesting an extracellular rather than intracellular function of prostatic TGase and polyamines. The observation that Km-values of rat prostate TGase for all three polyamines (57-120 microM, using N,N-dimethylated casein as protein substrate) were well below seminal polyamine concentrations is compatible with a regulatory role of polyamines in the process of seminal clot formation.

Cinnamoyl Inhibitors of Tissue Transglutaminase

Transglutaminases (TGases) catalyze the intermolecular cross-linking of certain proteins and tissue TGases (TG2) are involved in diverse biological processes. Unregulated, high TGase activities have been implicated in several physiological disorders, but few reversible inhibitors of TG2 have been reported. Herein, we report the synthesis of a series of novel trans-cinammoyl derivatives, discovered to be potent inhibitors of guinea pig liver transglutaminase. The most effective inhibitors evaluated can be sorted into two subclasses: substituted cinnamoyl benzotriazolyl amides and the 3-(substituted cinnamoyl)pyridines, referred to more commonly as azachalcones. Kinetic evaluation of both of these subclasses revealed that they display reversible inhibition and are competitive with acyl donor TGase substrates at IC50 values as low as 18 microM. An analysis of structure-activity relationships within these series of inhibitors permitted the identification of potentially important binding interactions. Further testing of some of the most potent inhibitors demonstrated their selectivity for TG2 and their potential for further development.

Gel-forming ability of small scale mud carp (Cirrhiana microlepis) unwashed and washed mince as related to endogenous proteinases and transglutaminase activities

Gel-forming ability of small scale mud carp (Cirrhiana microlepis) mince and washed mince was investigated with respect to their proteinase and transglutaminase (TGase) activities. Proteinases in sarcoplasmic fluid showed the optimum activity at 65 °C and pH 9, whereas autolytic activity was maximum at 70 °C and pH 10, indicating the presence of heat-stable alkaline proteinases. When mince was washed with three volumes of water twice, TGase and proteinases were mainly removed in the first washing cycle, resulting in a decreased autolytic activity of washed mince. Breaking force of the single washed mince gels was greater than the twice washed mince and the unwashed mince gels (p<0.05). Pre-incubation of mince pastes at 40 °C for 1 h prior to cooking (90 °C/30 min) increased breaking force of all samples, particularly the single washed mince (p<0.05). This coincided with an increase of higher molecular weight polymers observed on SDS-PAGE. Washing did not completely eliminate proteinases as it was evident by an increased trichloroacetic acid (TCA)-soluble oligopeptides of washed gels pre-incubated at 55 °C. Whiteness values of washed mince gels were greater than that of mince gels.

Enhanced Expression of Two Novel GTP-Independent Alternatively Spliced Forms of Tissue Transglutaminase in Human Platelets and Leukocytes.

Following vascular injury, blood coagulation and platelet activation trigger the wound healing process. The migration of blood cells through the leaky vessels, formation of new blood vessels, and the synthesis of extracellular matrix (ECM) are essential for tissue repair. Tissue transglutaminase (TTG) is a unique extracellular and intracellular enzyme that stabilizes tissues, binds and releases nitric oxide (NO), and hydrolyzes GTP and ATP. TTG's crosslinking (TGase) activity makes ECM resistant to protease digestion and aids wound healing. Recent studies demonstrate TTG binds and releases NO inhibiting platelet aggregation and neutrophil migration which inhibiting the inflammatory response. TTG is an enzyme with 687 amino acid residues. The active site involved in protein crosslinking is located at Cys277, while GTP/ATP binding domain is located in the N- and C-terminus and there are 18 free Cys-SH groups distributed throughout the molecule to bind NO. When vascular tissues derived from different phases of wound healing were analyzed on immunoblotting, we detected full-length and truncated forms of TTG antigens. To determine whether the truncated forms of TTG were formed by protein proteolysis or alternative splicing, we screened a human smooth muscle cell lambda cDNA library using a full-length human TTG cDNA as a probe. DNA sequencing analysis of the positive clones revealed that, in additional to wild type, two C-terminal truncated forms, TTG3 and TTG4, were also present. TTG3 and TTG4 were produced by a rare alternate splicing event utilizing alternate 5′ and 3′ splice site, located within exons XII and XIII, respectively. TTG3 and TTG4 were composed of 674 and 646 amino acid residues that shared identical N-terminal 622 amino acids with TTG with distinct 52 and 23 amino acids at the C-terminus that translated into proteins with the predicted Mr of 75 and 70 KDa, respectively. Structure-function studies using purified enzymes demonstrated that TTG3 and TTG4 showed the same calcium requirement as TTG, but had only 9 and 8% of residual TGase activity, respectively. TGase activity of TTG was inhibited by GTP with an IC50 of 6 microM, while both isoforms were not inhibited by up to 400 microM of GTP. GTP also failed to induce a conformational change in the molecule and both isoforms were proteolyzed by tyrpsin while full-length TTG remained intact. Both isoforms retained GTPase and ATPase activities. RT-PCR and immunoblotting demonstrated that TTG3 and TTG4 were expressed at less than 10 and 5% of TTG and were localized in the nucleus in human umbilical vascular endothelial (HUVEC) and vascular smooth muscle (VSMC) cells. In contrast, human leukocytes and platelets contained ~7-fold higher levels of both isoforms than TTG. In conclusion, we identified two novel C-terminal truncated forms of TTG that are expressed by HUVEC, VSMC, human leukocytes and platelets. This is the first report of the expression of two novel TTG isoforms in human blood cells. The different affinity for GTP and TGase activities, distinct intracellular localization and high expression levels in human leukocytes and platelets suggest a unique physiological function of these isoforms during hemostasis. In addition, TTG3 has two additional Cys-SH groups which could bind NO. The physiological significance of both isoforms of TTG in regulating wound repair is currently under investigation.

TGF-β1 up-regulates transglutaminase two and fibronectin in dermal fibroblasts: a possible mechanism for the stabilization of tissue inflammation

Transglutaminase (TGase) has been reported to stabilize tissue inflammation via the mediation of the polymerization of extracellular matrix proteins. A set of cytokines has been implicated in wound healing processes in the dermis. This study was undertaken in order to evaluate the effects of these cytokines on the expression of TGase 2 in human dermal fibroblasts (hDFs), in that TGase 2 is known to be the principal TGase in the dermis. In Western blot analysis, TGF-beta1 (1 ng/ml) treatment was found to steadily up-regulate TGase 2 expression for up to 7 days. However, such increases were not observed when the cells were treated with IL-1beta, IL-2, and TNF-alpha. In the enzyme assay, total TGase activities were closely related to the levels of TGase 2 expression. TGase 2 mRNA expression was up-regulated as the result of TGF-beta treatment in competitive RT-PCR. In the denatured SDS-PAGE, TGF-beta1 treatment resulted in marked induction of an approximately 220 kDa protein, which was revealed to be a fibronectin (FN) via western immunoblotting with an anti-FN antibody. Next, when the hDFs were treated with TGF-beta1 (1 ng/ml), FN expression was induced beginning at the third day after treatment. The immunoprecipitants generated by anti-FN antibody were positive for the anti-TGase 2 antibody, and the immune complexes were identified at molecular weights of 92 kDa. Collectively, TGF-beta1 stimulates the polymerization of FN via the action of TGase 2, which is supposed to to be an important mechanism in the stabilization of the inflammatory dermis.

Chronic ultraviolet radiation modulates epidermal differentiation as it up‐regulates transglutaminase 1 and its substrates

Ultraviolet radiation (UVR) stimulates cellular mitosis, which leads to epidermal hyperplasia. On the basis of hypothesis that chronic UVR may modulate differentiation as well as epidermal hyperplasia, we evaluated the modulation of markers of epidermal differentiation, such as transglutaminase 1 (TGase 1), filaggrin and loricrin, by chronic UVR in vivo. Total TGase activities assay or in situ TGase activities were measured in human and mouse skin. TGase 1 expression was identified by immunohistochemical staining in human skin. In the human, the pre-auricular skin of face was used for samples of chronic UVR, and the post-auricular skin was selected as non-UVR control. The changes of filaggrin and loricrin were identified by western immunoblots. In human and mouse epidermis, chronic UVR induced the increase of in situ TGase activities or total TGase activities as it up-regulated TGase 1 expression in the epidermis. As the substrates of TGase 1, chronic UVR induced the up-regulation of filaggrin and loricrin in mouse epidermis as well. At the same time, chronic UVR induced the marked epidermal hyperplasia in human and mouse skin. Chronic UVR stimulates epidermal differentiation as it up-regulates TGase 1 and its substrates. The modified epidermal differentiation is balanced with epidermal hyperplasia, leading to the maintenance of epidermal homeostasis in the UV-irradiated epidermis.

AtPng1p. The first plant transglutaminase.

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.

Formation of fibrinogen-based hydrogels using phototriggerable diplasmalogen liposomes.

We report the triggered release of Ca2+ from liposomal compartments to induce rapid gelation of protein-based hydrogels. Phototriggerable liposomes were designed by entrapping CaCl(2) within liposomes composed of 38:57:5 diplasmenylcholine (DPPlsC):disteroylphosphatidylcholine (DSPC):bacteriochlorophyll (Bchl). These liposomes release >80% of their entrapped Ca2+ within 15 min when irradiated at 800 nm (800 mW/cm2). A precursor solution, containing liposomes suspended in aqueous human fibrinogen and transglutaminase (TGase), remained fluid for several hours in the dark, but gelled rapidly when exposed to 800 nm excitation, as a result of photosensitized Ca2+ release and TG-induced fibrinogen cross-linking. TGase and hrFXIII activities, determined using a fluorimetric dansylcadaverine assay, were found to depend strongly on irradiation time and reaction temperature. SDS-PAGE of the photolyzed reaction mixture revealed that gelation arises from enzyme-catalyzed cross-linking of predominately the alpha and gamma chains of fibrinogen. This approach to the phototriggered formation of protein hydrogels creates new opportunities for biomaterials applications in drug delivery, tissue engineering, and wound healing.

GTP Binding and Signaling by Gh/Transglutaminase II Involves Distinct Residues in a Unique GTP-binding Pocket

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).

The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism

Rooting of wild-type tobacco ( Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation.

Calreticulin down-regulates both GTP binding and transglutaminase activities of transglutaminase II.

Enzyme regulation is an important mechanism for controlling cell proliferation and differentiation in response to extracellular signaling molecules. We have previously reported that a approximately 50 kDa protein (termed Gbetah) consistently copurified with Galphah (transglutaminase II, TGII) and that Gbetah down-regulates the GTPase function of TGII by associating with GDP-bound TGII [Baek et al. (1996) Biochemistry 35, 2651-2657]. In this study, we examined the identity of Gbetah by partial amino acid sequencing and immunological characterizations. The results strongly suggest that Gbetah is a protein known as calreticulin (CRT). When the regulatory role of CRT in the GTPase activity of TGII was examined, CRT inhibited GTP (GTPgammaS) binding and hydrolysis in a concentration-dependent manner. Moreover, CRT interacted only with GDP-bound TGII. These results demonstrate that CRT down-regulates the GTPase activity of TGII by associating with GDP-bound TGII. Studies on the modulation of the TGase activity of TGII revealed that CRT also inhibited TGase activity. The inhibition showed the two characteristics depend on guanine nucleotides occupying the GTPase active site. The inhibition of the "empty" form of the GTPase active site increased the Ca2+ requirement without changing the Vmax. On the other hand, the inhibition of the GDP-bound form decreased Vmax, but did not alter the Ca2+ requirement. Moreover, the GTPgammaS-bound TGII was virtually resistant to Ca2+-mediated stimulation of the TGase activity, indicating that the GTP-bound TGII does not function as a TGase. We concluded that CRT is the regulatory protein of TGII that down-regulates both GTPase and TGase activities, opposing the activators of TGII function.

OCCURRENCE OF TRANSGLUTAMINASE IN GREY MULLET (MUGIL CEPHALUS) MUSCLE AND IT'S EFFECT ON MINCED FISH PRODUCT

The gel strength of the minced fish product made from the unwashed mullet mince was found higher than that of the washed product. Transglutaminase (TGase) was found in the water soluble fraction of mullet muscle. It's optimal temperature was at 30C to 35C and the optimal pH was 7.5 to 7.7, Increased setting time at 30C, increased the gel strength of the minced fish product produced from the unwashed mullet mince. p-Chloromercuribenzoate (PCMB), an inhibitor to TGase was added to the mullet mince up to 2.0 mM, the mince was set at 35C and then heated to 85C. The gel strength of the product was inversely correlated to the PCMB concentration, Y 1 = - 0.45X 1 + 1.66 (r = 0.95). Studies of setting of the mullet mince at temperatures ranging 20 to 60C indicated that maximal gel strength was obtained at 35C, coinciding with the optimal temperature of TGase. Meat from 21.5 month-old grey mullet produced a minced product of higher gel strength than those produced from younger fish, although the muscle TGase activities were not significantly different.

Increase in transglutaminase and its extracellular products in response to an inflammatory stimulus by lipopolysaccharide.

Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. epsilon (gamma-Glutamyl)lysine and [3H]putrescine-labelled gamma-glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its gamma-glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.

Histamine in Mammary Gland: Pregnancy and Lactation

Abstract 1. Histamine concentration, histidine decarboxylase (HDC), histamine N-methyltransferase (HMT) and transglutaminase (TG-ase) activities have been followed in mouse and guinea pig mammary gland during pregnancy and lactation. 2. The most significant changes concerned histidine decarboxylase (HDC) activity and their pattern was similar for the two species. In either species 2nd trimester of pregnancy was associated with stimulation whereas early lactation was associated with reduction of the tissue HDC activity. 3. Hormonal control of HDC is suggested on the basis of variations in the enzyme activity and histamine concentration in mammary gland during estrous cycle. 4. The circumstantial evidence is provided indicating that histamine might be involved in pregnancy associated growth of mammary gland and in excretory activity of lactating gland.

Biological functions of the unusual guanine nucleotide-binding protein Gαh: transglutaminase II

Regulation of cellular response to external stimuli, hormones and neurotransmitters, is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins and their associated proteins. A new class of GTP-binding protein, Gαh is a bifunctional enzyme possessing two biological functions, namely GTPase and transglutaminase (TGase) activity. This bifunctional Gαh mediates the α1B-adrenoceptor signal to an 85- kDa phospholipase C-δ1 (PLC-δ1) and associates with a -50 kDa protein (Gβh) which regulates the the GTP/GDP binding affinity of Gαh. Gh, the holoenzyme is thus a heterodimer, and the subunits dissociate from each other upon activation with GTP or its analogues. The GTPase and TGase activities of Gαh are regulated by two reciprocal activators, Ca2+ and GTP. The GTP-binding subunit, Gαh directly interacts with the α1B-adrenoceptor and stimulates PLC-δ1. Gβh, however, neither interacts with the receptor nor stimulates PLC-δ1. Therefore, Gβh functions as a counterpart to the receptor for the activation of Gαh.