Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

Partial purification of γ-glutamyltransferase from human brain microvessels

Two forms of gamma-glutamyltransferase from human brain cortex microvessels were partially purified by gel permeation and ion-exchange and group-affinity chromatography. The specific activity of the purified preparations was 320-fold (detergent form) and 830-fold (proteolytic form) higher than that of the enzyme in the brain cortex homogenate. The relative molecular mass of the proteolytic form of the enzyme was about 90,000 as determined by gel permeation chromatography. The major part of the enzyme (about 80%) was absorbed on Con A-Sepharose 4B. The pH optima for transfer reactions with -glutamyl-4-nitroanilide as donor and glycylglycine andl-cystine as acceptors were in the range of 8.2 to 9.0. The studied enzyme was inhibited by a mixture ofl-serine and borate and by bromcresol green.     

Substrate Preference of γ-Glutamyltransferase from Caps of Fruiting Bodies of Lentinus edodes

Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000–9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.     

Solubilization and some properties of γ-glutamyltransferase from human brain microvessels

Detergents Triton X-100, sodium deoxycholate, and octyl--D-glucopyranoside, and proteinase papain proved to be excellent agents solubilizing the -glutamyl-transferase (-GT) from human brain cortex microvessels. Ficin also solubilized -GT but to a lesser extent than papain. The relative molecular mass of the detergent-solubilized enzyme form was greater than 200,000 (in the presence of Triton X-100). The relative molecular mass of the proteinase-solubilized form was slightly greater than that of albumine. -GTs of microvessels from five human brain regions and from the choroid plexus were tested for their specificity toward acceptors. The best acceptors were found to be (in decreasing order of activity)l-cystine, glycylglycine,l-glutamine,l-methionine, andl-alanine. The findings suggest that the main features of -GT of the human blood-brain barrier are very similar to those of -GTs from other human tissues.     

Ganoellipsic acids A–C,lanostane triterpenoids from artificially cultivated fruiting bodies of Ganoderma ellipsoideum

Three undescribed lanostane triterpenoids, ganoellipsic acids A–C (1–3), together with seven known Ganoderma lanostanoids (4–10), were isolated from artificially cultivated fruiting bodies of Ganoderma ellipsoideum, strain BCC 16634. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. The C-25 absolute configuration of 1 was determined to be 25S using the phenylglycine methyl ester (PGME) method.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

Crystal Structure of γ-Hexachlorocyclohexane Dehydrochlorinase LinA from Sphingobium japonicum UT26

LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ hexachlorocyclohexane (HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene via γ-pentachlorocyclohexene. We determined the crystal structure of LinA at 2.25 Å by single anomalous dispersion. LinA exists as a homotrimer, and each protomer forms a cone-shaped α + β barrel fold. The C-terminal region of LinA is extended to the neighboring subunit, unlike that of scytalone dehydratase from Magnaporthe grisea, which is one of the most structurally similar proteins identified by the DALI server. The structure we obtained in this study is in open form, in which γ-HCH can enter the active site. There is a hydrophobic cavity inside the barrel fold, and the active site is largely surrounded by the side chains of K20, L21, V24, D25, W42, L64, F68, C71, H73, V94, L96, I109, F113, and R129. H73 was considered to function as a base that abstracts the proton of γ-HCH through its interaction with D25. Docking simulations with γ-HCH and γ-pentachlorocyclohexene suggest that 11 residues (K20, I44, L64, V94, L96, I109, A111, F113, A131, C132, and T133) are involved in the binding of these compounds and support the degradation mechanism.     

Low concentrations of reactive γ-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation

Oxidative stress has been strongly implicated in pathological processes. Isoketals are highly reactive γ-ketoaldehydes of the isoprostanes pathway of free radical-induced peroxidation of arachidonic acid that are analogous to cyclooxygenase-derived levuglandins. Because aldehydes, that are much less reactive than isoketals, have been shown to trigger platelet activation, we investigated the effect of one isoketal (E₂-IsoK) on platelet aggregation. Isoketal potentiated aggregation and the formation of thromboxane B₂ in platelets challenged with collagen at a concentration as low as 1 nM. Moreover, the potentiating effect of 1 nM isoketal on collagen-induced platelet aggregation was prevented by pyridoxamine, an effective scavenger of γ-ketoaldehydes. Furthermore, we provide evidence for the involvement of p38 mitogen-activated protein kinase in isoketal-mediated platelet priming, suggesting that isoketals may act upstream the activation of collagen-induced cytosolic phospholipase A₂. Additionally, the incubation of platelets with 1 nM isoketal led to the phosphorylation of cytosolic phospholipase A₂. The cytosolic phopholipase A₂ inhibitors AACOCF3 and MAFP both fully prevented the increase in isoketal-mediated platelet aggregation challenged with collagen. These results indicate that isoketals could play an important role in platelet hyperfunction observed in pathological states such as atherosclerosis and thrombosis through the activation of the endogenous arachidonic acid cascade.     

Studies of γ-glutamyltransferase activity in the brain tissue post mortem

Our experiments showed that the activity of -glutamyltransferase (-GT) did not remarkably change in homogenates of mouse, rat, and bovine brains during the first four days post mortem. In the course of that period, the brain microvessels also retained their -GT activity. -GT of microvessels from bovine brain cortex, solubilized with sodium deoxycholate, was eluted in the void volume V_o when chromatographed on a Sephadex G-200 column with the detergent Triton X-100. In human post mortem brains, the specific activity of -GT in choroid plexi was found to be about five times higher than that in the cerebral cortex, white matter, basal ganglia, pons, and cerebellum but about four times lower than that in the microvessels obtained from the studied brain regions. Our findings suggest that it is possible to study the components of the blood-brain barrier on material from deceased subjects.     

Sulfation of (1→3)-β-d-glucan from the fruiting bodies of Russula virescens and antitumor activities of the modifiers

A water-insoluble (1→3)-β-d-glucan isolated from the fresh fruiting bodies of Russula virescens was sulfated using sulfur trioxide-pyridine complex as reagent in dimethyl sulfoxide. Depending on the reaction conditions, the products showed different degrees of sulfation (DS) ranging from 0.17 to 1.17 and different weight average molecular weights (M_ws) ranging from 2.5 × 10⁴ to 1.2 × 10⁵ Da. Moreover, the antitumor activities of the five sulfated derivatives against Sarcoma 180 tumor cell were tested both in vitro and in vivo. The results indicated that the native (1→3)-β-d-glucan did not show antitumor activity, while the sulfated derivatives exhibited enhanced antitumor activities. This study demonstrated that DS and M_w could influence the antitumor activities of the sulfated derivatives.     

Exploring the unfolding mechanism of γ-glutamyltranspeptidases: The case of the thermophilic enzyme from Geobacillus thermodenitrificans

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.     

α-(1 → 3)-D-glucans from fruiting bodies of selected macromycetes fungi and the biological activity of their carboxymethylated products

Purpose of work  

To show biological activity of carboxymethylated α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi on human tumor and normal cells.     

Differential regulation of γ-glutamyltransferase mRNAs in four human tumour cell lines

Human γ-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-α (TNF-α), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-α and TPA did not alter these parameters. In LNCap cells, TNF-α, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-α, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.     

Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

5′-Monohydroxyphylloquinone from Anacystis and Euglena

The monohydroxy analogue of phylloquinone found in Anacystis nidulans and Euglena gracilis has been characterized as 5′-monohydroxyphylloquinone by MS analysis.