Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of⁷⁵Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of⁷⁵Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria

Summary We have studied the 3-mercaptopyruvate pathway (transamination pathway) ofl-cysteine metabolism in rat liver mitochondria.l-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. Whenl-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withl-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation froml-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatl-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.     

Inhibition of sulfate-forming activity in rat liver mitochondria by (aminooxy)acetate

Summary The effect of (aminooxy)acetate, an inhibitor of aminotransferases, on the sulfate formation froml-cysteine andl-cysteinesulfinate in rat liver mitochondria was studied. Incubation of 10 mMl-cysteine with rat liver mitochondria at 37°C in the presence of 10 mM 2-oxoglutarate and 10 mM glutathione resulted in the formation of 4.60 and 1.52µmol of sulfate and thiosulfate, respectively, per 60 min per mitochondria obtained from 1 g of liver. Under the same conditions sulfate formation froml-cysteinesulfinate was 24.96µmol, but thiosulfate was not formed. The addition of (aminooxy)acetate at 2 mM or more completely inhibited the sulfate and thiosulfate formation froml-cysteine and the sulfate formation froml-cysteinesulfinate. These findings support our previous conclusion that cysteine transamination and 3-mercaptopyruvate pathway (MP pathway) are involved in the sulfate formation froml-cysteine in rat liver mitochondria (Ubuka et al., 1992).     

Effect ofN-acetylcysteine administration on cysteine and glutathione contents in liver and kidney and in perfused liver of intact and diethyl maleate-treated rats

Summary Effect ofN-acetyl-l-cysteine (NAC) administration on cysteine and glutathione (GSH) contents in rat liver and kidney was studied using intact and diethyl maleate (DEM)-treated rats and perfused rat liver. Cysteine contents increased rapidly, reaching peak at 10 min after intraperitoneal NAC administration. In liver mitochondria it increased slowly, reaching peak at 60 min. GSH content did not change significantly in these tissues. However, in liver and kidney depleted of GSH with DEM, NAC administration restored GSH contents in 60 and 120 min, respectively. Perfusion with 10 mM NAC resulted in 76% increase in liver cysteine content, but not in GSH content. Liver perfusion of DEM-injected rats with 10 mM NAC restored GSH content by 15%. Present findings indicate that NAC is an effective precursor of cysteine in the intact liver and kidney and in the perfused rat liver, and that NAC stimulated GSH synthesis in GSH-depleted tissues.     

Cystathionine γ-lyase contributes to selenomethionine detoxification and cytosolic glutathione peroxidase biosynthesis in mouse liver

We earlier found that seleno-l-methionine (L-SeMet) as a food source of selenium (Se) is directly converted to methylselenol (CH₃SeH), α-ketobutyrate, and ammonia by the mouse hepatic cystathionine γ-lyase. The purpose of this study was to clarify the biological role of cystathionine γ-lyase in Se detoxification and cytosolic glutathione peroxidase (cGPx) biosynthesis because another metabolic pathway to CH₃SeH via seleno-l-cystathionine and seleno-l-cysteine (l-SeCyH) from l-SeMet has been shown by several enzymatic reactions. When mice were treated with either toxic doses of l-SeMet or a Se-deficient diet, the cystathionine γ-lyase activity for l-SeMet was invariable, suggesting that this enzyme was effective in both detoxification and biotransformation of Se. Concerning Se biotransformation into cGPx, production of H₂Se as the possible precursor was not observed by the in vitro reaction of the liver cytosol with CH₃SeH. When l-SeMet was administered at the nutritional dose to mice fed a Se-deficient diet, levels of both cGPx mRNA and cGPx protein were significantly restored. This recovery was not comparatively suppressed by coadministration of periodate-oxidized adenosine, an inhibitor of S-adenosylhomo-cystenase, where the conversion of l-SeMet to l-SeCyH is inhibited. However, the recovery was strongly suppressed when propargylglycine, an inhibitor of cystathioine γ-lyase that catalyzes the α,γ-elimination reaction of both l-SeMet and seleno-l-cystathionine, was treated. These results suggest that cystathionine γ-lyase is a notable enzyme, in SeMet metabolism and that CH₃SeH produced by the enzymatic reaction is utilized for cGPx biosynthesis.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Astrocytes as potential modulators of mercuric chloride neurotoxicity

Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca²⁺-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.     

Protective role of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid on antioxidant defense system in erythrocytes of albino rats exposed to nickel sulfate

In this experimental study, we investigated whether l-ascorbic acid has any influence on the blood antioxidant defense system, lipid peroxidation and hematological parameters of the albino rats exposed to nickel sulfate(NiSO₄).Twenty four adult rats were divided into four groups of six animals in each group. The control rats were untreated and comprised Group I. Group II rats were administered nickel sulfate (2.0 mg/100 g b.wt.; intraperitonially, i.p.). Group II rats were treated orally l-ascorbic acid (50 mg/100 g b.wt.) and Group IV rats were given both nickel sulfate and l-ascorbic acid simultaneously on alternate days until the tenth dose. The hematological parameters were assessed: red blood corpuscle counts, packed cell volume %, hemoglobin concentration, white blood corpuscle counts and platelets count decreased significantly and clotting time increased significantly in nickel treated rats. We also observed increase malondialdehyde (MDA) and decrease glutathione level (GSH) in erythrocytes of nickel treated rats. The activities of erythrocyte antioxidant enzymes like superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were significantly increased in rats treated with nickel sulfate. Simultaneously treatment of l-ascorbic acid exhibited a possible protective role on the toxic effect of nickel sulfate on the hematological values, erythrocyte MDA and GSH concentrations as well as antioxidant enzymatic defense system.     

Effects of Z-FA.FMK on d-galactosamine/tumor necrosis factor-alpha-induced kidney injury and oxidative stress in mice

Background The aim of this investigation was to demonstrate that benzyloxicarbonyl-l-phenylalanyl-alanine-fluoromethylketone (Z-FA.FMK), which is a pharmacological inhibitor of cathepsin B, has protective role on the kidney injury that occurs together with liver injury. Methods BALB/c male mice used in this study were divided into four groups. The first group was given physiologic saline only, the second group was administered Z-FA.FMK alone, the third group received d-galactosamine and tumor necrosis factor-alpha (d-GalN/TNF-α), and the fourth group was given both d-GalN/TNF-α and Z-FA.FMK. One hour after administration of 8 mg/kg Z-FA.FMK by intravenous injection, d-GalN (700 mg/kg) and TNF-α (15 μg/kg) were given by intraperitoneal injection. Results In the group given d-GalN/TNF-α, the following results were found: severe degenerative morphological changes in the kidney tissue, a significant increase in the number of activated caspase-3-positive tubular epithelial cell, an insignificant increase in the number of proliferating cell nuclear antigen (PCNA)-positive tubular epithelial cell, a decrease in the kidney glutathione (GSH) levels, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, an increase in the kidney lipid peroxidation (LPO) levels, lactate dehydrogenase (LDH) activity, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities, uric acid and urea levels. In contrast, in the group given d-GalN/TNF-α and Z-FA.FMK, a significant decrease in the d-GalN/TNF-α-induced degenerative changes, a decrease in the number of activated caspase-3-positive tubular epithelial cell, a insignificant decrease in the number of PCNA-positive tubular epithelial cell, an increase in the kidney GSH levels, CAT, SOD and GPx activities, a decrease in the kidney LPO levels, LDH activity, serum AST and ALT activities, uric acid and urea levels were determined. Conclusion These results suggest that pretreatment with Z-FA.FMK markedly lessens the degree of impairment seen in d-GalN/TNF-α-induced kidney injury, which occurred together with liver injury in mice.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K _m = 2.2 M;V _max = 23 nmol·(g FW)^–1·h^–1]; (ii) a low-affinity, high-capacity component [K _m = 159 M;V _max = 742 nmol·(g FW)^–1·h^–1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)^–1 h^–1];·mM^–1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K _m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K _i = 1–5 M; and a low affinity (K _i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.Abbreviations AEC S-2-aminoethyl-l-cysteine - K _i equilibrium constant - WT wild-type     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl -cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R) reagents where R is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding -S-(-aminoethanethiol) and -S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization

λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (M_r) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg²⁺. With 0.2mM Cd²⁺ or Zn²⁺, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (M_r 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent K_m values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (K_i=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell. Dedicated to Professor A. Prison on the occasion of his 80th birthday     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.