Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.     

Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification

A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch.     

Identification of a high affinity leukotriene C4-binding protein in rat liver cytosol as glutathione S-transferase

A soluble high affinity binding unit for leukotriene (LT) C4 in the high speed supernatant of rat liver homogenate was characterized at 4 degrees C as having a single type of saturable affinity site with a dissociation constant of 0.77 +/- 0.27 nM (mean +/- S.E., n = 5). The binding activity was identified as the liver cytosolic subunit 1 (Ya) of glutathione S-transferase, commonly known as ligandin, by co-purification with the catalytic activity during DEAE-cellulose column chromatography and 11,12,14,15-tetrahydro-LTC4 (LTC2)-affinity gel column chromatography; resolution into two major bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Mr 23,000 and 25,000, of which only the smaller protein was labeled with [3H]LTC4 coupled via a photoaffinity cross-linking reagent; and immunodiffusion analysis with rabbit antiserum to glutathione S-transferase which showed a line of identity between the purified LTC4-binding protein and rat liver glutathione S-transferase. The affinity-purified binding protein bound 800 pmol of [3H] LTC4/mg of protein and possessed 12 mumol/min/mg of glutathione transferase activity as assayed with 1-chloro-2,4-dinitrobenzene as substrate. The enzyme activity of the cytosolic LTC4-binding protein was inhibited by submicromolar quantities of unlabeled LTC4, and the binding activity for [3H]LTC4 was blocked by the ligandin substrates, hematin and bilirubin. The high affinity interaction between LTC4 and glutathione S-transferase suggests that glutathione S-transferase may have a role in LTC4 disposition and that previous studies of LTC4 binding to putative receptors in nonresponsive tissues may require redefinition of the binding unit.     

Eosinophil peroxidase oxidation of thiocyanate. Characterization of major reaction products and a potential sulfhydryl-targeted cytotoxicity system

Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.     

Biochemical and immunological characterization of rat spleen prostaglandin D synthetase

Rat spleen prostaglandin D synthetase (Christ-Hazelhof, E., and Nugteren, D. H. (1979) Biochim. Biophys. Acta 572, 43-51) is very similar to rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi O. (1985) J. Biol. Chem. 260, 12410-12415) as judged by their pI (4.7-5.2), Mr (26,000-27,000), and self-inactivation during the isomerase reaction from prostaglandin H2 to prostaglandin D2. However, the amino acid compositions of these two enzymes were quite different. Furthermore, the spleen enzyme was associated with the glutathione S-transferase activity, differing from the brain enzyme. The synthetase and transferase activities of the spleen enzyme showed almost identical pH and glutathione dependencies, the optimum pH = 8.0 and Km for glutathione = 300 microM. The Km values for prostaglandin H2 and 1-chloro-2,4-dinitrobenzene (a substrate for the transferase) were about 200 microM and 5 mM, respectively. The synthetase activity was dose-dependently inhibited by 1-chloro-2,4-dinitrobenzene (IC50: approximately 5 mM) and more strongly by nonsubstrate ligands, such as bilirubin and indocyanine green (IC50: 150 and 2 microM, respectively). Both the synthetase and transferase activities of the purified enzyme dose-dependently decreased and showed identical immunotitration curves by incubation with antibody against this enzyme, but remained unchanged when treated with antibody against the brain enzyme. The antibody specific for the spleen enzyme absorbed almost all of the synthetase activity and about 10% of the transferase activity in the spleen, but not the transferase activity in the liver, heart, and testis. These results show that the two types of prostaglandin D synthetase are similar but different enzymes and that the spleen enzyme is a unique glutathione S-transferase differing from other isozymes and their subunits reported previously.     

Fatty acid binding protein is a major determinant of hepatic pharmacokinetics of palmitate and its metabolites

Disposition kinetics of [(3)H]palmitate and its low-molecular-weight metabolites in perfused rat livers were studied using the multiple-indicator dilution technique, a selective assay for [(3)H]palmitate and its low-molecular-weight metabolites, and several physiologically based pharmacokinetic models. The level of liver fatty acid binding protein (L-FABP), other intrahepatic binding proteins (microsomal protein, albumin, and glutathione S-transferase) and the outflow profiles of [(3)H]palmitate and metabolites were measured in four experimental groups of rats: 1) males; 2) clofibrate-treated males; 3) females; and 4) pregnant females. A slow-diffusion/bound model was found to better describe the hepatic disposition of unchanged [(3)H]palmitate than other pharmacokinetic models. The L-FABP levels followed the order: pregnant female > clofibrate-treated male > female > male. Levels of other intrahepatic proteins did not differ significantly. The hepatic extraction ratio and mean transit time for unchanged palmitate, as well as the production of low-molecular-weight metabolites of palmitate and their retention in the liver, increased with increasing L-FABP levels. Palmitate metabolic clearance, permeability-surface area product, retention of palmitate by the liver, and cytoplasmic diffusion constant for unchanged [(3)H]palmitate also increased with increasing L-FABP levels. It is concluded that the variability in hepatic pharmacokinetics of unchanged [(3)H]palmitate and its low-molecular-weight metabolites in perfused rat livers is related to levels of L-FABP and not those of other intrahepatic proteins.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

Human platelets that had been preincubated with 5-hydroxy[(3)H]tryptamine and [(32)P]P(i) were stirred with various agents; the secretion of 5-hydroxy[(3)H]tryptamine from platelet granules and the radioactivity of platelet [(32)P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[(3)H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E(1), which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[(3)H]tryptamine. Prostaglandin E(1) also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca(2+) concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714-722; (1978) Thromb. Res.12, 619-628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E(1) may initiate processes that decrease the Ca(2+) concentration in the cytosol, so inhibiting both the Ca(2+)-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E(1) did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca(2+) concentration in the platelet cytosol. As prostaglandin E(1) did inhibit the release of 5-hydroxy[(3)H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.     

Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A(2)s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation.

We have recently reported that members of the heparin-binding group II subfamily of secretory PLA(2)s (sPLA(2)s) (types IIA and V), when transfected into 293 cells, released [(3)H]arachidonic acid (AA) preferentially in response to interleukin-1 (IL-1) and acted as "signaling" PLA(2)s that were functionally coupled with prostaglandin biosynthesis. Here we show that these group II subfamily sPLA(2)s and the type X sPLA(2) behave in a different manner, the former being more efficiently coupled with the prostaglandin-biosynthetic pathway than the latter, in 293 transfectants. Type X sPLA(2), which bound only minimally to cell surface proteoglycans, augmented the release of both [(3)H]AA and [(3)H]oleic acid in the presence of serum but not IL-1. Both types IIA and V sPLA(2), the AA released by which was efficiently converted to prostaglandin E(2), markedly augmented IL-1-induced expression of cyclooxygenase (COX)-2 in a heparin-sensitive fashion, whereas type X sPLA(2) lacked the ability to augment COX-2 expression, thereby exhibiting the poor prostaglandin E(2)-biosynthetic response unless either of the COX isozymes was forcibly introduced into type X sPLA(2)-expressing cells. Implication of phospholipid scramblase, an enzyme responsible for the perturbation of plasma membrane asymmetry, revealed that the scramblase-transfected cells became more sensitive to types IIA and V, but not X, sPLA(2), releasing both [(3)H]AA and [(3)H]oleic acid in an IL-1-independent manner. Thus, although phospholipid scramblase-mediated alteration in plasma membrane asymmetry actually led to the increased cellular susceptibility to the group II subfamily of sPLA(2)s, several lines of evidence suggest that it does not entirely mimic their actions on cells after IL-1 signaling. Interestingly, coexpression of type IIA or V, but not X, sPLA(2) and phospholipid scramblase resulted in a marked reduction in cell growth, revealing an unexplored antiproliferative aspect of particular classes of sPLA(2).     

Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [(3)H]GTP and [(3)H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.     

Evidence of luminal phosphatidylcholine secretion in rat ileum

BACKGROUND: Intestinal mucus not only facilitates substrate absorption, but also forms a hydrophobic, phosphatidylcholine (PC) enriched, barrier against luminal gut contents. METHODS: For evaluation of the origin of PC in intestinal mucus, we first analyzed the mucus PC in mice with absent biliary phospholipid secretion (mdr2 (-/-) mice) using electrospray ionization (ESI) tandem mass spectroscopy (MS/MS). Second, in situ perfused rat jejunum, ileum and colon were analyzed after i.v. bolus injections of 155 pmol [(3)H]-PC. Additional in vitro experiments were performed with isolated mucosal cells after incubation with the PC precursor [(3)H]-choline. RESULTS: In mdr2 (-/-) mice and control animals no significant quantitative difference in mucus PC was found, indicating that mucus PC is of intestinal and not biliary origin. In situ perfusion studies detected intestinal secretion of [(3)H]-PC, which was stimulated in presence of 2 mM taurocholate (TC). Secretion rates of [(3)H]-PC were highest in ileum (9.0+/-0.8 fmol h(-1)xcm(-1)), lower in jejunum (4.3+/-0.5) and minimal in colon (0.8+/-0.2). It compares to an intestinal secretion of native PC originating to 64% from bile, 9% from jejunum, 28% from ileum, and 1% from colon. Complementary in vitro studies showed 30-min secretion rates for [(3)H]-PC to be highest in enterocytes from ileum (26.5+/-5.3% of intracellular [(3)H]-PC) and jejunum (19.8+/-2.9%), and significantly lower in colonocytes (8.4+/-1.3%). CONCLUSION: PC in the intestinal mucus originates from secretion by ileal and jejunal enterocytes.     

Synthesis of 2-bromomethyl-3-hydroxy-2-hydroxymethyl-propyl pyrimidine and theophylline nucleosides under microwave irradiation. Evaluation of their activity against hepatitis B virus

Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b-d as well as theophylline (7) with 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) under microwave irradiation gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-2-methyl-thio pyrmidin-4(1H)-ones 3a-d and 7-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides 1,1 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis[(2-(methylthio)-pyrimidin-4(1H)-ones] 4a-c, and 7,7 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). The deacetylated derivatives were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV with mild cytotoxicity.     

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).     

Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200 microM, with a kmax of 0.111 min-1 and KI = 185 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Identification of Tyr115 labeled by S-(4-bromo-2,3-dioxobutyl)glutathione in the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 3-3.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 3-3 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. The kobs exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 900 microM, with a kmax of 0.073 min-1 and KI = 120 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, completely protects against inactivation by S-BDB-G. About 2.0 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated concomitant with 100% inactivation, whereas only 0.96 mol of reagent/mol subunit is incorporated in the presence of S-hexylglutathione when activity is fully retained. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH4, reacted with N-ethylmaleimide, and digested with trypsin. Analysis of the tryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Tyr115 as the amino acid whose reaction with S-BDB-G correlates with inactivation. Examination of the stability of S-(4-bromo-2,3-dioxobutyl)glutathione and modified enzyme in the absence and presence of dithiothreitol and under acidic conditions suggests that for stable linkage to peptides, the carbonyl moieties of the reagent should be reduced immediately after modification of a protein. Comparison of results from the 4-4 and 3-3 isoenzymes of rat liver glutathione S-transferase (both of the mu gene class) indicates: the 4-4 isoenzyme exhibits a greater affinity for S-BDB-G; Cys86 is labeled by [3H]S-BDB-G in both isoenzymes but is nonessential for activity; in the 3-3 isoenzyme, Cys86 is more accessible to S-BDB-G; and Tyr115 is an important residue in the hydrophobic binding site of both enzymes.     

Bioavailability of starch and postprandial changes in splanchnic glucose metabolism in pigs

Changes in splanchnic metabolism in pigs were assessed after meals containing slowly or rapidly digested starch. The pigs were fed a mixed meal containing a "slow" native (n = 5) or a "rapid" pregelatinized (n = 5) cornstarch naturally enriched with [(13)C]glucose. Absorption of [(13)C]glucose was monitored by the arteriovenous difference technique, and infusion of D-[6, 6-(2)H(2)]glucose in the jugular vein was used to calculate the systemic appearance of [(13)C]glucose. Arteriovenous balance data obtained during a 12-h study period showed that the fraction of ingested glucose equivalent appearing as glucose in the portal vein was 49.7 +/- 7.2% for the slow starch and 48.2 +/- 7.5% for the rapid starch (P = 0.86). These values, corrected for the gut extraction of circulating [(13)C]glucose, became 66.4 +/- 5.6 and 65. 3 +/- 5.6%, respectively (P = 0.35). Isotope dilution data indicated that systemic appearance of exogenous [(13)C]glucose represented 62. 9 +/- 7.6 and 67.4 +/- 3.0% of the oral load for slow and rapid starch, respectively (P = 0.68). Arterial glucose utilization by the gut increased from 7.3 +/- 0.9 micromol x kg(-1) x min(-1) before the meal to 8.5 +/- 1.6 micromol x kg(-1) x min(-1) during absorption, independently of the nature of the starch. Thus splanchnic glucose metabolism was unaffected by the nature of starch ingested.