Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Isolation and purification of multiple forms of γ-glutamyl transpeptidase from rat brain

Four different forms of the enzyme -glutamyl transpeptidase were isolated from rat brain by chromatography on concanavalin A. An approximate 1500-fold purification was achieved. The four forms were characterized with respect to molecular weight,K _m for -glutamyl-p-nitroanilide, mobility on polyacrylamide gels, and inhibitory effects of borate-serine. The multiple forms of the enzyme were found to have molecular weights ranging from 74,000 to 234,000 andK _ms of 0.07 to 8.6 mM. It was determined that in brain, the major portion of the enzyme activity is associated with plasma membrane fragments and endoplasmic reticulum.     

Purification and characterization of a novel thermophilic β-galactosidase from Picrophilus torridus of potential industrial application

Extremophiles - Intracellular β-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE...     

A fluorometric assay for γ-glutamyl transpeptidase: Demonstration of enzymatic activity in cultured cells of neural origin

A new fluorometric assay was developed for the measurement of -glutamyl transpeptidase (-GTP). The assay utilizes as a substrate the synthetic compound 7--glutamylamido-4-methyl coumarin which is cleaved by -GTP to yield the highly fluorescent product 7-amino-4-methyl coumarin. Optimal excitation and emission wavelengths for the assay are 345 nm and 470 nm, respectively, and the sensitivity of the assay is greatly enhanced by the high-pressure liquid chromatographic separation of the product from the substrate. The assay is minimally 25 times more sensitive than the conventional spectrophotometric assay and permits analysis of as little as 5000 cultured cells of neuronal and glial origin. Analysis of a variety of cultured cells of neuronal and glial origin with this assay suggests that -GTP is largely present in glia and to a lesser extent in neurons.     

Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl₂ or MgCl₂. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl₂, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.     

Conformational changes and stabilization induced by phosphate binding to 5′-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.     

Relationship between kinetic properties of γ-glutamyl transpeptidase and the structure of its saccharide moiety

Gamma-glutamyl transpeptidase (EC 2.3.2.2; GGT) is a plasma-membrane bound glycoenzyme, the saccharide moiety of which is rather heterogeneous and organ specific. It has been stated that GGT catalyses three types of reactions, i.e., hydrolysis, transpeptidation and autotranspeptidation. The initial velocity equation, involving all these reactions, is shown in the present report. Mathematical analysis of the equation resulted in a. definition of the constant of half saturation (K_hs). The value of K_hs was used for characterization of kinetics of GGT from rat organs differing in the structure of GGT oligosaccharide chains. No significant organ differences were found, when the K_hs values of GGT from the brain, kidney and pancreas equalled 0.61 mM, 0.68 mM and 0.68 mM, respectively. On the contrary, when two different glycoforms of GGT from the pancreas were compared, distinct values of K_hs were obtained (l.43 mM and 0.67 mM, respectively). It is therefore being suggested that the saccharide chains of GGT are involved in its kinetic properties. However, this effect is masked when the enzyme, non-fractionated into glycoforms, is analysed, even though the saccharide moiety is specific for the organ studied.     

Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ-glutamyl transpeptidase is mainly glutathione degradation-dependent

Helicobacter (H.) suis is the most prevalent non-H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of γ-glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT-treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT-mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H(2)O(2) concentrations, generated in a cell-independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGT-mediated generation of pro-oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate.     

γ-Glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region

Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned, expressed in pET-28-E. coli expression system as a heterodimeric enzyme with molecular weights of 45 and 20 kDa for large and small subunit, respectively. It was purified by nickel affinity chromatography with hydrolytic and transpeptidase activity of 1.82 U/mg and 4.35 U/mg, respectively. Sequence analysis revealed that GGTBP was most closely related to Bacillus licheniformis GGT and had all the catalytic residues and nucleophiles for autoprocessing recognized from E. coli. It was optimally active at pH 8 and 60°C. It exhibited pH stability from pH 6-9 and high thermostability with t(1/2) of 15 min at 70°C. It had K(m), V(max) of 0.045 mM, 4.35 μmol/mg/min, respectively. Decoupling of autoprocessing by co-expressing large and small subunit in pET-Duet1-E. coli expression system yielded active enzyme with transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto 95 aa did not affect autoprocessing of GGT however activity was lost with truncation beyond 63 aa.     

Defined subcomplexes of the A1 ATPase from the archaeon Methanosarcina mazei Gö1: biochemical properties and redox regulation

The potential A(1) ATPase genes ahaA, ahaB, ahaC, ahaD, ahaE, ahaF, and ahaG from the anaerobic archaeon Methanosarcina mazei G?1 were overexpressed in Escherichia coli DK8 (pTL2). An A(1) complex was purified to apparent homogeneity and shown by Western blot and N-terminal sequence analyses to contain subunits A, B, C, D, and F but to be devoid of subunits E and G. Further removal of subunit C was without effect on ATPase activity. The enzyme was most active at pH 5.2 and required bisulfite and acetate for maximal activity. Kinetic studies confirmed three new inhibitors for A(1) ATPases (diethylstilbestrol and its derivatives hexestrol and dienestrol) and identified redox modulation as a new type of regulation of archaeal A(1) ATPases.     

Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble γ-glutamyl transpeptidase

Background  

In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions.     

Membrane-bound γ-glutamyl transpeptidase and its rôle in phenylalanine absorption-reabsorption in the larva of Musca domestica

Most of the γ-glutamyl transpeptidase (γ-GTP) activity of actively feeding third instar housefly larvae is located on the brush border of the proximal half of the Malpighian tubules and the brush border of epithelial cells of the anterior and posterior portions of the midgut. It is concluded that these membranes are the major sites of synthesis of the dipeptide, γ-l-glutamyl-l-phenyl-alanine (γ-glu-phe).In effect, γ-GTP and γ-glu-phe form a highly specific system for the absorption and reabsorption of phenylalanine from the lumen of the midgut and Malpighian tubules. Thus, membrane-bound γ-GTP combines with phenylalanine and glutathione and the resulting γ-glu-phe is translocated across the cell membrane and released within the cell. The dipeptide then enters the blood, presumably by simple diffusion in response to the concentration gradient generated by its build-up within the cell. It accumulates in the blood during larval growth and finally is consumed upon the onset of puparium tanning.Puparium formation was accompanied by an abrupt, ecdysone-induced appearance of intense γ-GTP activity on the epidermal cell membrane at the epidermis-cuticle interface. Epidermal cell γ-GTP activity was maximal 1 to 2 hr after puparium formation, after which time it began to diminish rapidly. It became virtually undectectable by the larval-pupal apolysis. Functionally, this hormonally induced new γ-GTP may catalyse reaction(s) which result in a rapid liberation of phenylalanine from γ-glu-phe for its subsequent conversion to tyrosine and quinones for tanning the puparium.The possibility that γ-GTP may also function in the transport of other amino acids in the housefly and in other insect genera is considered in terms of the Orlowski-Meister concept of a ‘γ-glutamyl cycle’.     

γ-Glutamyl transpeptidase: catalytic,structural and functional aspects

Summary -Glutamyl transpeptidase catalyzes transfer of the -glutamyl moiety of glutathione to amino acids, dipeptides, and to glutathione itself; the enzyme also catalyzes the hydrolysis of glutathione to glutamate and cysteinyl-glycine. This review deals with the tissue distribution and localization of the enzyme in mammals, the catalytic properties of the enzyme (including its inhibition by reversible and irreversible inhibitors), structural studies on the enzyme, and new findings about its physiological function.     

Direct evidence for inter-organ transport of glutathione and that the non-filtration renal mechanism for glutathione utilization involves γ-glutamyl transpeptidase

A direct examination of the inter-organ cycle of glutathione metabolism was made by determining glutathione levels in plasma obtained from various blood vessels of the rat. High levels of GSH were found in hepatic vein plasma, relative to arterial and systemic venous levels, reflecting translocation of GSH from the liver to the plasma. Renal vein plasma has a level that is 20% of arterial plasma indicating that the kidney removes glutathione from plasma not only by glomerular filtration (which can account for 20–30% of the glutathione removed), but also by a non-filtration mechanism. Inhibitors of γ-glutamyl transpeptidase decrease the fraction of glutathione removed by the kidney to a value approaching that filtered, indicating that the non-filtration mechanism involves γ-glutamyl transpeptidase.     

A Highlights from MBoC Selection: αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.     

Potent and selective inhibitors of PI3Kδ: obtaining isoform selectivity from the affinity pocket and tryptophan shelf

A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.     

Docking studies on kinesin spindle protein inhibitors: an important cooperative ‘minor binding pocket’ which increases the binding affinity significantly

Fifteen KSP inhibitors were docked into the receptor and the binding mode was analyzed for the first time. It was considered that in addition to the main binding pocket all the inhibitors merged in, there exists a cooperative minor binding pocket, which could be explored for significantly increased binding affinity. In addition, a good linear relationship between the biological activities and the lowest binding free energies has also been found. This may help in predicting the binding affinity of newly designed KSP inhibitors. Figure Two binding pockets considered after the analysis. Seven docked ligands (compound 1–7) were overlapped at the binding site. All inhibitors tested interacted with the main pocket, while CK0106023, interacted also with the cooperative minor pocket mainly surrounded by Arg221 and Ala218. Coloring of the binding site surface are different ends of each amino acid residue: blue represents amino group while red means carboxyl     

Enterobacter soli sp. nov.: A Lignin-Degrading ��-Proteobacteria Isolated from Soil

A Gram-negative bacterium that formed cream-colored colonies designated strain LF7 was isolated from soil collected in the Tambopata National Reserve in Madre de Dios, Peru. 16S rRNA sequence comparisons indicate that LF7 is a novel Enterobacter sp. closely related to E. asburiae JCM 6051(T) [AB004744] and E. aerogenes JCM 1235(T) [AB004750] based on their sequence homologies (p-distance: 1.06 and 1.19%, respectively). DNA G + C content was 52.8 mol% which is within the range reported for E. asburiae (55-57 mol%). The major cellular fatty acids present in the LF7 strain were C(16:0) (27.3%), C(16:1) ω7c and/or C(16:1) ω6c (16.3%), C(18:1) ω7c (16.1%), C(17:0) cyclo (12.4%), C(14:0) 3-OH and/or C(16:1) iso-I (8.9%), C(14:0) (7.6%), C(12:0) (3.9%), C(17:0) (2.4%), C(13:0) 3-OH and/or C(15:1) iso-H (1.7%), C(13:0) (1.1%), and C(18:2) ω6,9c and/or C(18:0) ante (0.5%). The cellular fatty acid profile, G + C content, phenotypic and biochemical characteristics were consistent with its placement in the genus Enterobacter. The name Enterobacter soli is proposed for this bacterium.