Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K_m value for reduced glutathione was 11 μM for both GGT1 and GGT5. However, the K_m values for oxidized glutathione were 9 μM for GGT1 and 43 μM for GGT5. Our data show that the K_m values for leukotriene C₄ are equivalent for GGT1 and GGT5 at 10.8 and 10.2 μM, respectively. This assay was also used to evaluate serine–borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.     

Purification and partial characterization of the glutathione S-transferase of rat erythrocytes

The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (M_r 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of M_r 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The K_m values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I₅₀) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (K_i = 8 μM) and glutathione (K_is = 4 μM; K_ii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (K_i = 5 μM).     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Inhibition of 17β-hydroxysteroid dehydrogenase (17β-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17β-HSD activities. Microsomal 17β-HSD was inhibited by C₁₈ -,C₁₉- and C₂₁-steroids. Soluble 17β-HSD was highly specific for C₁₈-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (K_I=0.3 μM) and danazol (K_I=0.6 μM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17β-HSD.K_I-values for estradiol-17β, (0.8 μM), progesterone (27.0 μM) and 20α-dihydroprogesterone (1.5 μM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C₁₈-, C₁₉-, and C₂₁-steroids for the active site of microsomal 17β-HSD.     

Hydrolysis and transfer reactions catalyzed by gamma-glutamyl transpeptidase; evidence for separate substrate sites and for high affinity of L-cystine.

γ-Glutamyl transpeptidase was studied with L- and D-γ-glutamyl-p-nitroanilide as γ-glutamyl donors. No autotranspeptidation occurred with the D-γ-glutamyl donor or when the L-γ-glutamyl donor was used at concentrations lower than 10 μM. The K_m values for hydrolysis were 5 and 31 μM for the L- and D-γ-glutamyl donors, respectively; the corresponding V_max values were identical. The γ-glutamyl donor site of the enzyme thus exhibits low stereospecificity (but high affinity), while the acceptor site exhibits absolute L-specificity. The K_m value for L-cystine as acceptor is very low (30 μM); the same value was obtained with L- and D-γ-glutamyl donors. The data are consistent with a ping pong mechanism and the existence of separate donor and acceptor enzyme sites. The present findings thus extend the usefulness of γ-glutamyl-p-nitro-anilide as a substrate in probing the catalytic behavior of this enzyme.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones as urease inhibitors

A comparative kinetic study of the inhibition of urea hydrolysis by 9 substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones (BI I–IX) has been carried out. The inhibition had reversible competitive mode; the inhibition constants K _i, varied from 29 to 754 μM in dependence of structure of BI I–IX. Three BI I–IX, characterized by the K _i values ranged from 29 to 82 μM, may be used as potential therapeutic agents in gastroenterology for treatment of gastroduodenal ulcers.     

Ascorbate-2-phosphate inhibition of ascorbate-2-sulfate sulfohydrolase from bovine liver.

The hydrolysis of ascorbate-2-sulfate by the enzyme, ascorbate-2-sulfate sulfohydrolase, purified from bovine liver has been shown to be powerfully inhibited by ascorbate-2-phosphate. The inhibition by ascorbate phosphate is competitive with a K_I of 0.3 μM. Na₂HPO₄ also inhibits by an apparent non-competitive process. The Na₂HPO₄ concentration at 50% inhibition is 7.7 μM. A possible control role for ascorbate phosphate in ascorbate biochemistry is suggested.     

Km (CO2) values of ribulose-1,5-bisphosphate carboxylase in grasses of different C4 type

Statistical analysis of K_m (CO₂) values of ribulose-1,5-bisphosphate (RuBP) carboxylase from 35 C₄ grass species shows that the mean value for PEP-carboxykinase (PCK) type C₄ species (41.4±s.e. 2.2 μM CO₂) is significantly different from that of NAD-malic enzyme (NAD-ME) type species (55.3±3.1 μM CO₂) or NADP-malic enzyme (NADP-ME type species (52.5±s.e. 2.0μM CO₂). These C₄ type differences remain detectable within both the eu-panicoid and chloridoid grass subfamilies. By contrast, no between-subfamily differences were found within C₄ types. Variation in K_m (CO₂) values of RuBP carboxylase may be related to in vivo differences in CO₂ concentration at the enzyme site, mediated perhaps by differences in CO₂-leakiness of C₄ leaf ‘photosynthetic carbon reduction’ (PCR or ‘Kranz’) tissue.     

Metabolism of leukotrienes

Summary The in vitro metabolism of leukotriene B₄ is initiated by -hydroxylation. This reaction is followed by oxidation of the -hydroxyl group to a carboxyl group. In vivo extensive -oxidation occurs and the main excreted products after administration of leukotriene B₄ are water and carbon dioxide.Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal y-glutamyl residue, catalyzed by the enzyme -glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E₄, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C₄. In rats, the two major fecal metabolities of leukotriene C₄4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E₄. These compounds are formed in reactions between leukotriene E₄ or 11-trans leukotriene E₄ and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.     

C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C₂-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O₂⁻) production. The preincubation of PMN for 15 min with C₂-ceramide increased by up to almost 3-fold the amounts of O₂⁻ generated in response to 0.1 and 1 μM fMLP. Priming was detected at C₂-ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C₂-ceramide, C₆-ceramide (N-hexanoylsphingosine) could prime for O₂⁻ generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O₂⁻ generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O₂⁻ production; however, the combination of TNF-α and C₂-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C₂-ceramide and TNF-α. C₂-ceramide at 2 μM did not enhance the production of O₂⁻ induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B₄ (LTB₄), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C₂-ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A₂ or D (PLC, PLA₂, PLD) were involved in the priming effect by C₂-ceramide. However, C₂-ceramide inhibited in a dose-related manner the production of O₂⁻ induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C₂-ceramide. The priming O₂⁻ production by C₂-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C₂-ceramide.     

The effects of tetrahydroisoquinolinecarboxylic acids on tyrosine 3-monooxygenase

The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor (K_i = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor (K_i = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid (K_i = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid (K_i = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro, decreases in the K_i of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the K_i of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC₅₀ = 100 μm) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo.     

Leukotriene A4-induced bronchoconstriction in the guinea pig in vivo

Administration of leukotriene A₄ (0.03 - 0.3 μg kg^?1 i.v.) to anesthetized spontaneously breathing guinea pigs produced pronounced changes in pulmonary resistance, dynamic compliance and blood pressure. The pulmonary responses were unaffected either by pretreatment with indomethacin or following desensitization to leukotriene B₄ but were significantly attenuated by the leukotriene D₄ receptor antagonist, FPL-55712. Following administration of leukotriene A₄ increased levels of leukotriene C₄-immunoreactive material were determined in the plasma and neutrophil accumulation was observed in the lung. It was concluded that leukotriene A₄ induced bronchoconstriction in the guinea pig either by acting directly on the leukotriene D₄ receptor site or more probably through efficient metabolism in the lung to peptido-lipid leukotrienes which in turn exerted direct bronchoconstrictor actions.     

Diguanosine 5′,5‴ -P1,P4-tetraphosphate causes specific inhibition and desensitization of Artemia trypsin-like proteinase in the hydrolysis of a high-affinity,arginine-rich substrate

Diguanosine tetraphosphate (Gp₄G) inhibits Artemia trypsin-like proteinase. The inhibition is quite specific and presents a K_i about 1 μM. Gp₄G modifies both maximum velocity and affinity of the proteinase. The proteinase, without losing activity, can be desentsitized to the inhibition by Gp₄G at low concentrations (1–10 μM). Gp₄G inhibits the hydrolysis of the high-affinity, arginine-rich substrate, protamine, to a lesser extent that of polylysine and does not affect the catalysis of other basic proteins. The results may suggest the involvement of Gp₄G in the regulation of the hydrolysis of polypeptides containing clusters of arginine.     

Glutathione-degrading enzymes of microvillus membranes

Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Pharmacological comparison of L-serine borate and glutathione as inhibitors of metabolism of LTC4 to LTD4 by the isolated guinea pig trachea

Cumulative dose-response curyes to leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD)₄ were obtained on indomethacin (5 μM) treated isolated guinea pig tracheal spiral strips. LTC₄ curves, in the presence of either glutathione (GSH; 10 mM) or L-serine borate (SB; 45 mM), were not antagonized by FPL-55712 (3 μM), a selective LTD₄ receptor antagonist. LTC₄ curves on trachea treated with a lower concentration of GSH (1 mM), and LTD₄ curves were competitively antagonized by FPL-55712. LTC, curves on GSH (10 mM) treated trachea were 2 fold to the left of those on SB treated tissues. This effect of GSH was blocked by pretreatment with nordihydro-guiaretic acid (30 μM), an inhibitor of 5-lipoxygenase.GSH (10 μM) and SB (45 mM) are effective inhibitors of conversion of LTC₄ into functionally important levels of LTD₄ by the guinea pig trachea. In addition, GSH appeares to enhance LTC₄ responsiveness by increasing synthesis of a contractile 5-lipoxygenase product(s), possibly LTC₄. From the data it is suggested that for inhibition of LTC₄ metabolism, SB may be more usefull when examining responses to exogenously applied LTC₄, while GSH (10 mM) may be useful when examining responses to endogenously generated LTC₄.     

Potentiating effects of pertussis toxin on leukotriene C4 induced formation of inositol phosphate and prostacyclin in human umbilical vein endothelial cells

Leukotriene C₄ is an arachidonic acid metabolite and an important mediator of inflammation and anaphylaxis that is known to induce production of prostacyclin in endothelial cells. The goal of this study was to examine the signal transduction mechanisms activated by leukotriene C₄ stimulation. Formation of inositol phosphates was measured to determine the activation of phospholipase C and pertussis toxin was used to explore the role of G-proteins. Additionally, we evaluated the role of protein kinase C in these events, especially whether there was an interaction between pertussis toxin mediated effects and the activity of protein kinase C. Leukotriene C₄ induced a dose- and time-dependent formation of inositol phosphates and prostacyclin. The response to leukotriene C₄ was greater than the response to leukotriene D₄ even after treatment with L-serine borate complex, suggesting the presence of a specific leukotriene C₄ receptor. Exposure to pertussis toxin potentiated, time-dependently, the leukotriene C₄ induced formation of inositol phosphates and prostacyclin through a mechanism which was altered by manipulation of protein kinase C activity. The exact mechanism is not clear but our results are consistent with a postulated dual mechanism of phospholipase C control, in which leukotriene C₄ induced stimulation is attenuated by a pertussis toxin sensitive G-protein. J. Cell. Physiol. 177:103–108, 1998. © 1998 Wiley-Liss, Inc.     

Metabolism of leukotriene A4 into C4 by human platelets

Tritium-labelled leukotriene A4 is converted by a suspension of human platelets into leukotriene C4. The conversion is stimulated by reduced glutathione and is dependent on the platelet concentration. Formation of leukotriene C4 is temperature and time dependent and is destroyed by heating the platelets at 100 degrees C for 5 min. Verification of leukotriene C4 formation was obtained by conversion into leukotriene D4 during reaction of the HPLC-purified platelet-derived leukotriene C4 with commercial gamma-glutamyl transpeptidase. In separate experiments we incubated authentic tritiated leukotriene C4 with human platelets and we showed the formation of tritiated leukotriene D4, demonstrating the presence of gamma-glutamyl transpeptidase activity in these cells. This activity could be blocked by the presence of reduced glutathione in the incubation mixture. In contrast, erythrocytes converted tritiated leukotriene A4 almost exclusively into leukotriene B4. Although platelets have been reported to lack 5-lipoxygenase activity, our study demonstrates that platelets possess the necessary machinery to transform leukotriene A4 into leukotrienes C4 and D4. Our results suggest that an intracellular interaction between platelets and leukotriene A4-forming cells, e.g., polymorphonuclear leukocytes, could lead to the formation of these potent peptidolipids in the circulation.