Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.     

Selective elution of rodent glutathione S-transferases and glyoxalase I from the S-hexyglutathione-Sepharose affinity matrix.

1. The major hepatic glutathione S-transferases (GSTs) from gerbil, guinea-pig, hamster, mouse and rat comprise Ya- (Mr 25,500-25,800), Yb- (Mr 26,100-26,400), Yc- (Mr 27,000-27,500) and Yf- (Mr 24,800) type subunits. 2. In all rodent species the GST subunits possess characteristic affinities for S-hexyglutathione-Sepharose and are eluted at distinct positions when a gradient of counter-ligand is employed to develop this affinity gel. The enzymes that bind to this matrix can be eluted, according to their subunit composition, in the order Ya-, Yc-, Yf- and Yb-containing GST; glyoxalase I, also retained by S-hexylglutathione-Sepharose, is eluted after the major GST YbYb peak. 3. Conditions are also described for the isocratic affinity elution of S-hexylglutathione-Sepharose that allow rat GST to be divided into four separate fractions (pools 1-4). A further fraction (pool 5) can be prepared from material that does not bind S-hexylglutathione-Sepharose and is obtained by chromatography on glutathione-Sepharose. 4. The sequential use of S-hexylglutathione-Sepharose and glutathione-Sepharose has facilitated the isolation of novel GSTs by enriching the various affinity-purified fractions with different subunits. This strategy allowed the Yk (Mr 25,000) and Yo (Mr 26,500) subunits from rat testis as well as Y1 (Mr 25,700) from rat kidney to be rapidly purified. 5. The binding properties of GST subunits for S-hexylglutathione-Sepharose have been compared with their Km values for GSH. The elution order from this matrix is inversely related to the Km value. The GSTs that do not bind to S-hexylglutathione-Sepharose have considerably higher Km values for GSH (i.e. greater than 2.0 mM) than do those enzymes that readily bind to the affinity gel (i.e. 0.13-0.77 mM). GST YkYk and YoYo, which have weak affinities for S-hexylglutathione-Sepharose, possess intermediate Km values for GSH of 1.0 and 1.2 mM respectively.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Human glutathione S-transferases. The Ha multigene family encodes products of different but overlapping substrate specificities

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5'-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.     

Lipoxygenase-another pathway for glutathione conjugation of xenobiotics: A study with human term placental lipoxygenase and ethacrynic acid.

In this study, we examined the ability of human term placental lipoxygenase (HTPLO) to catalyze glutathione (GSH) conjugate formation from ethacrynic acid (EA) in the presence of linoleic acid (LA) and GSH. HTPLO purified by affinity chromatography was used in all the experiments. The results indicate that the process of EA-SG is enzymatic in nature. The reaction shows dependence on pH, the enzyme, and the concentration of GSH, LA, and EA. The optimal assay conditions to observe a maximal rate of EA-SG formation required the presence of 0.3 mM LA, 0.2 mM EA, 2.0 mM GSH, and approximately 300 microg HTPLO in the reaction medium buffered at pH 9.0. Under the experimental conditions employed, the reaction exhibited K(m) values of 1.1 mM, 200 microM, and 130 microM for GSH, LA, and EA, respectively. The estimated specific activity of HTPLO-catalyzed EA-GS formation was approximately 4.4 +/- 0.4 micromol/min/mg protein. This rate is more than twofold greater than the rate noted for the reaction mediated by the purified human term placental glutathione transferase. Under physiologically relevant conditions (20 microM LA, 2.0 mM GSH, at pH 7.4), HTPLO produced EA-SG at 56% of the maximal rate noted under optimal assay conditions. Nordihydroguaiaretic acid, the classical inhibitor of different lipoxygenases, significantly blocked the reaction. It is proposed that free radicals are involved in the process of EA-SG formation by HTPLO. The evidence gathered in this in vitro study suggests for the first time that lipoxygenase present in the human term placenta is capable of EA-SG formation.     

H.p.l.c. separation and study of the charge isomers of human placental glutathione transferase.

Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo.     

Intrabiliary glutathione hydrolysis. A source of glutamate in bile

High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.     

The glutathione S-transferase activity in the kidney of rainbow trout (Salmo gairdneri)

Trout kidney contains 2.3 mmol GSH/kg. The cytosolic glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate is 0.35 mumol/min/mg protein. There is no detectable activity with 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, p-nitrobenzyl chloride or p-nitrophenyl acetate. A variable proportion of the activity does not bind to a glutathione-affinity matrix. Its Km values for GSH and 1-chloro-2,4-dinitrobenzene are 3.0 and 5.1 mM, respectively. The rest of the activity is eluted from the affinity matrix as one main and two minor peaks. The main peak has Km values for GSH and 1-chloro-2,4-dinitrobenzene of 0.4 and 4.5 mM, respectively. Its subunit Mr is 22,900. The activity in the main peak is inhibited progressively by 1-chloro-2,4-dinitrobenzene with a rate constant of 0.11/min.     

Purification and properties of acetyl-CoA:L-glutamate N-acetyltransferase from human liver.

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km,acetyl-CoA 4.4 mM, Ki,acetyl-CoA 4.7 mM, Km,glutamate 8.1 mM, Ki,glutamate 8.8 mM; for the products, Ki,acetylglutamate 0.28 mM, Ki,CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.     

Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction.

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Ketopantoate hydroxymethyltransferase. II. Physical, catalytic, and regulatory properties.

Some physical, catalytic, and regulatory properties of ketopantoate hydroxymethyltransferase (5,10-methylenetetrahydrofolate: alpha-ketoisovalerate hydroxymethyltranferase) from Escherichia coli are described. This enzyme catalyzes the reversible synthesis of ketopantoate (Reaction 1), an essential precursor of pantothenic acid. (1) HC(CH3)2COCOO- + 5,10-methylene tetrahydrofolate f in equilibrium r HOCH2C(CH3)2COCOO- + tetrahydrofolate It has a molecular weight by sedimentation equilibrium of 255,000, a sedimentation coefficient (S20,w) of 11 S, a partial specific volume of 0.74 ml/g, an isoelectric point of 4.4, and an absorbance, (see article), of 0.85. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid analyses give a subunit molecular weight of 27,000 and 25,700, respectively; both procedures indicate the presence of 10 identical subunits. The NH2-terminal sequence is Met-Tyr---. The enzyme is stable and active over a broad pH range, with an optimum from 7.0 to 7.6. It requires Mg2+ for activity; Mn2+, Co2+, Zn2+ are progressively less active. The enzyme is not inactivated by borohydride reduction in the presence of excess substrates, i.e. it is a Class II aldolase. Reaction 1f is partially inhibited by concentrations of formaldehyde (0.8 mM) and tetrahydrofolate (0.38 mM) below or near the Km values, apparent Km values are 0.18, 1.1 and 5.9 mM for tetrahydrofolate, alpha-ketoisovalerate, and formaldehyde, respectively. For Reaction 1r, apparent Km values are 0.16 and 0.18 mM, respectively, for ketopantoate and tetrahydrofolate, and the saturation curves for both substrates show positive cooperativity. Forward and reverse reactions occur at similar maximum velocities (Vmax approximately equal to 8 mumol of ketopantoate formed or decomposed per min per mg of enzyme at 37 degrees). Only 1-tetrahydrofolate is active in Reaction 1; d-tetrahydrofolate, folate, and methotrexate were neither active nor inhibitory. However, 1-tetrahydrofolate was effectively replaced with conjugates containing 1 to 6 additional glutamate residues; of these, tetrahydropterolpenta-, tetra-, and triglutamate were effective at lower concentrations than tetrahydrofolate itself; they were also the predominant conjugates of tetrahydrofolate present in E. coli. Alpha-Ketobutyrate, alpha-ketovalerate, and alpha-keto-beta-methylvalerate replaced alpha-ketoisovalerate as substrates; pyruvate was inactive as a substrate, but like isovalerate, 3-methyl-2-butanone and D- or L-valine, inhibited Reaction 1. the transferase has regulatory properties expected of an enzyme catalyzing the first committed step in a biosynthetic pathway. Pantoate (greater than or equal to 500 muM) and coenzyme A (above 1 mM) all inhibit; the Vmax is decreased, Km is increased, and the cooperativity for substrate (ketopantoate) is enhanced. Catalytic activity of the transferase is thus regulated by the products of the reaction path of which it is one component; transferase synthesis is not repressed by growth in the presence of pantothenate.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

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.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

化学合成虎纹捕鸟蛛毒素-I基因的克隆和表达

本文报道了全化学合成虎纹捕鸟蛛毒素－Ⅰ基因在大肠杆菌中的表达,表达产物为Ｎ－端是谷胱甘肽硫转移酶的融合蛋白．经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化,凝血酶酶解融合蛋白,得到重组ＨＷＴＸ－Ⅰ（ｒＨＷＴＸ－Ⅰ）．质谱和氨基酸顺序分析均表明ｒＨＷＴＸ－Ⅰ系正确表达产物．还原复性的ｒＨＷＴＸ－Ⅰ表现出与天然ＨＷＴＸ－Ⅰ生物学活性的一致性．     

Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms

Although recent studies suggest involvement of glutathione transferase (GST) of blood vessels in vasodilation by nitroglycerin, GST forms in blood vessels remain to be studied. In this study, three GST forms (pI values 8.3, 6.6, and 4.8) were purified from human aorta and four (pI values 6.0, 5.6, 5.3, and 4.6) from the heart by affinity chromatography followed by chromatofocusing. The major form of both aorta (pI 4.8) and heart (pI 4.6) was identified as GST-pi, and the other five forms were immunologically related to GST-mu, suggesting that the five belong to the Mu class. Among nine human GST forms, including three in the Alpha class purified from the liver, GST-mu, aorta pI 8.3 form, and GST-I (a form of the Alpha class, corresponding to GST-epsilon (B1B1)) showed high activities toward nitroglycerin, 1.08, 0.85, and 0.78 units/mg protein, respectively. GST-pi did not exhibit the activity. The Km values of the aorta form (pI 8.3) for glutathione (GSH) and nitroglycerin were calculated as 0.12 and 1.1 mM, respectively. The Km values of GST-mu and GST-I for GSH were 0.29 and 0.09 mM, and those for nitroglycerin were 2.5 and 0.3 mM, respectively. The activity of the pI 8.3 form as well as GST-mu toward nitroglycerin was inhibited by bromosulfophthalein, which is known to inhibit the relaxation of rabbit aorta induced by nitroglycerin, at the lower concentration (IC50, 2 microM) than was GST-I (IC50, 32 microM). Two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis revealed that five forms in the Mu class are homo- or heterodimers of five different subunits named M1 (pI 7.0/Mr 27,000), M2 (6.6/27,000), M3 (6.0/27,000), N1 (6.5/26,500), and N2 (5.9/26,500). The subunit structures of the five forms are as follows: pI 8.3 form, M1M2; 6.6 form, M2N1; 6.0 form, M3M3; 5.6 form, M3N2; and 5.3 form, N2N2. M3 and N2 seem to correspond to the subunits of GST-mu, and -4 (Board, P. G., Suzuki, T., and Shaw, D. C. (1988) Biochim. Biophys. Acta 953, 214-217), respectively. These subunits except N1 are different from each other at two or three positions in the first 20 residues of N-terminal amino acid sequence. These results indicate the presence of five different subunits in the human Mu class and also suggest that GST-M1M2 and -M2N1 found in the aorta are involved in the expression of the pharmacologic effect of nitroglycerin.     

pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.