Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves

The monoterpene ketone l-menthone is specifically converted to l-menthol and l-menthyl acetate and to d-neomenthol and d-neomenthyl-beta-d-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves. The selectivity of product formation results from compartmentation of the menthol dehydrogenase with the acetyl transferase and that of the neomenthol dehydrogenase with the glucosyl transferase. Soluble enzyme preparations, but not particulate preparations, from mature peppermint leaves catalyzed the NADPH-dependent reduction of l-menthone to both epimeric alcohols, and the two dehydrogenases responsible for these stereospecific transformations were resolved by affinity chromatography on Mātrex Gel Red A. Both enzymes have a molecular weight of approximately 35,000, possess a K(m) for NADPH of about 2 x 10(-5)m, are very sensitive to inhibition by thiol-directed reagents, and are not readily reversible. The menthol dehydrogenase showed a pH optimum at 7.5, exhibited a K(m) for l-menthone of about 2.5 x 10(-4)m, and also reduced d-isomenthone to d-neoisomenthol. The neomenthol dehydrogenase showed a pH optimum at 7.6, exhibited a K(m) for l-menthone of about 2.2 x 10(-5)m, and also reduced d-isomenthone to d-isomenthol. These stereochemically distinct, but otherwise similar, enzymes are of key importance in determining the metabolic fate of menthone in peppermint, and they are probably typical of the class of dehydrogenases thought to be responsible for the metabolism of monoterpene ketones during plant development.     

Metabolism of Monoterpenes : Early Steps in the Metabolism of d-Neomenthyl-beta-d-Glucoside in Peppermint (Mentha piperita) Rhizomes

Previous studies have shown that the monoterpene ketone l-[G-³H] menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-β-d-glucoside which is then transported to the rhizome (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). Analysis of the disposition of l-[G-³H]menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome. Studies with d-[G-³H]neomenthyl-β-d-glucoside as substrate, using excised rhizomes or rhizome segments, confirmed the hydrolysis of the glucoside as an early step in metabolism at this site, and revealed that the terpenoid moiety was further converted to a series of ether-soluble, methanol-soluble, and water-soluble products. Studies with d-[G-³H]neomenthol as the substrate, using excised rhizomes, showed the subsequent metabolic steps to involve oxidation of the alcohol back to menthone, followed by an unusual lactonization reaction in which oxygen is inserted between the carbonyl carbon and the carbon bearing the isopropyl group, to afford 3,4-menthone lactone. The conversion of menthone to the lactone, and of the lactone to more polar products, were confirmed in vivo using l-[G-³H]menthone and l-[G-³H]-3,4-menthone lactone as substrates. Additional oxidation products were formed in vivo via the desaturation of labeled neomenthol and/or menthone, but none of these transformations appeared to lead to ring opening of the p-menthane skeleton. Each step in the main reaction sequence, from hydrolysis of neomenthyl glucoside to lactonization of menthone, was demonstrated in cell-free extracts from the rhizomes of flowering mint plants. The lactonization step is of particular significance in providing a means of cleaving the p-menthane ring to afford an acyclic carbon skeleton that can be further degraded by modifications of the well-known β-oxidation sequence.     

Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita)

(-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-)-menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone. Acid hydrolysis of the nonvolatile material released over half of the radioactivity to the steamvolatile fraction, and the major component of this fraction was identified as (+)-neomenthol by radiochromatographic analysis and by synthesis of crystalline derivatives, thus suggesting the presence of a neomenthyl glycoside. Thin layer chromatography, ion exchange chromatography, and gel permeation chromatography on Bio-Gel P-2 allowed the purification of the putative neomenthyl glycoside, and these results suggested that the glycoside contained a single, neutral sugar residue. Hydrolysis of the purified glycoside, followed by reduction of the resulting sugar moiety with NaB(3)H(4), generated a single labeled product that was subsequently identified as glucitol by radio gas-liquid chromatography of both the hexatrimethylsilyl ether and hexaacetate derivative, and by crystallization to constant specific radioactivity of both the alditol and the corresponding hexabenzoate. These results, along with studies on the hydrolysis of the glycoside by specific glycosidases, strongly suggest that (+)-neomenthyl-beta-d-glucoside is a major metabolite of (-)-menthone in flowering peppermint. This is the first report on the occurrence of a neomenthyl glycoside, and the first evidence implicating glycosylation as an early step in monoterpene catabolism.     

Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves

The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (—)-menthyl acetate, and leaf discs converted exogenous (—)-[G-³H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (—)-[G-³H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K_m and velocity for (—)-menthol were 0.3 mm and 16 nmol/hr· mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.     

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular.     

Variations in composition of peppermint oil in relation to production areas

Compositional data obtained for peppermint oil fromMentha piperita L. produced in Tasmania as well as oils produced in other major production areas, were analysed by principal coordinate analysis. The 6 variates included in the analysis were the oil compounds limonene, cineole, menthone, menthofuran, menthyl acetate and menthol. In general, Tasmanian oils were characterised by low menthofuran, low limonene, low menthyl acetate and to a lesser extent high menthone, low cineole and high menthol concentrations relative to most major production areas. When the variation in composition of oil samples from within Tasmania was displayed in 3 dimensions (using the first 3 principal coordinates) it was not possible to achieve any pronounced separation of oils produced at different locations within southern Tasmania. Principal coordinates which were based mainly on cineole and menthol concentrations, respectively, did allow a degree of separation between oils produced in southern and northern Tasmania. Generally, oils produced in northern Tasmania had lower cineole and, in some cases, higher menthol than southern Tasmanian oils. Oil extracted from regrowth herb after commercial harvest was distinguished by having very high menthyl acetate, low menthone, high menthol, high menthofuran, low cineole and low limonene concentrations. The effect of some cultural and environmental factors on oil composition is also discussed.     

Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species

The volatile oil of mature Mentha piperita (peppermint) leaves contains as major components the oxygenated p-menthane monoterpenes l-menthol (47%) and l-menthone (24%) as well as very low levels of the monoterpene olefins limonene (1%) and terpinolene (0.1%), which are considered to be probable precursors of the oxygenated derivatives. Immature leaves, which are actively synthesizing monoterpenes, produce an oil with comparatively higher levels of limonene (approximately 3%), and isolation of the pure olefin showed this compound to consist of approximately 80% of the l-(4S)-enantiomer and approximately 20% of the d-(4R)-enantiomer. The time course of incorporation of [U-14C]sucrose into the monoterpenes of M. piperita shoot tips was consistent with the initial formation of limonene and its subsequent conversion to menthone via pulegone. d,l-[9-3H]Limonene and [9,10-3H]terpinolene were prepared and tested directly as precursors of oxygenated p-menthane monoterpenes in M. piperita shoot tips. Limonene was readily incorporated into pulegone, menthone, and other oxygenated derivatives, whereas terpinolene was not appreciably incorporated into these compounds. Similarly, d,l-[9-3H]limonene was specifically incorporated into pulegone in Mentha pulegium and into the C-2-oxygenated derivative carvone in Mentha spicata, confirming the role of this olefin as the essential precursor of oxygenated p-menthane monoterpenes. Soluble enzyme preparations from the epidermis of immature M. piperita leaves converted the acyclic terpenoid precursor [1-3H]geranyl pyrophosphate to limonene as the major cyclic product, providing a further indication that this olefin plays a central role in the formation of oxygenated monoterpenes in Mentha. No free intermediates were detected in the cyclization of geranyl pyrophosphate to limonene, suggesting that the olefin is the first cyclic intermediate to arise in the pathway, and resolution of the biosynthetic limonene, by crystallization of the derived d- and l-carvoximes, indicated an enantiomer mixture nearly identical to that isolated from the leaf oil.     

Metabolism of monoterpenes: demonstration that (+)-cis-isopulegone, not piperitenone, is the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint (Mentha piperita)

Piperitenone is commonly considered to be the key intermediate in the conversion of (-)-isopiperitenone to (+)-pulegone in peppermint; however, [3H]piperitenone gave rise only to the inert metabolite (+)-piperitone when incubated with peppermint leaf discs. Under identical conditions, (-)-[3H]isopiperitenone was efficiently incorporated into (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and yielded an additional metabolite identified as (+)-cis-isopulegone; piperitenone was poorly labeled. Moreover, (+)-cis-[3H]isopulegone was rapidly converted to (+)-pulegone, (-)-menthone, and (+)-isomenthone in leaf discs, and the reduction of (+)-[3H]pulegone to (-)-menthone and (+)-isomenthone was similarly documented. Each step of the pathway was demonstrated in a crude soluble preparation from peppermint leaf epidermis and each of the relevant enzymes was partially purified in order to compare relative rates of catalysis. The results of these studies indicate that the endocyclic double bond of (-)-isopiperitenone is reduced to yield (+)-cis-isopulegone, which is isomerized to (+)-pulegone as the immediate precursor of (-)-menthone and (+)-isomenthone, and they rule out piperitenone as an intermediate of the pathway.     

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.     

Antibacterial properties and major bioactive components of Mentha piperita essential oils against bacterial fruit blotch of watermelon

Plant-derived essential oil is an alternative to antibiotics, eliminating the concern of developing antibiotic-resistant bacterial strains. In this study, using the half-divided Petri plate assays, 32 volatile essential oils were screened for their antibacterial activity against Acidovorax citrulli (Acc). Sweet basil and peppermint oils were the most effective against Acc, with subsequent trials showing that peppermint oil to be the most active. Using gas chromatography–mass spectrometry, the major compositions of peppermint oil were analysed. Among the various compositions of peppermint oil, menthol, neomenthol, isopulegone and 1,8-cineole were significantly active against Acc and each component at 0.2% concentration inhibited all bacterial growth. This study demonstrated in vitro and in vivo antibacterial activities of peppermint oil and its active components against Acc. These results suggest the use of peppermint oil as a potential antibacterial agent to treat seed with Acc.     

Two-phase system membrane reactor with cofactor recycling.

For the purpose of constructing a two-phase system reactor the enzymatic process of l-menthol production with cofactor recycling was studied as a model. The half-life of the menthone reductase immobilized onto activated carbon was 4 times as high as that of the free enzyme. The enzyme was capable of regenerating NADH when methyl isobutyl carbinol was used as a second substrate. Continuous production of l-menthol was achieved by using a reactor equipped with a hydrophobic microfiltration membrane. It was found that both NAD(H) and the enzyme could be retained in the reactor and the products, l-methanol and methyl isobutyl ketone, passed through the membrane. The production of l-methanol was maintained for 270 h at a rate of 46.1 g l-1 d-1, and had decreased by one-half at 607 h. The recycling number of NAD(H) was 2500 (max. 3020) during the operation. The number of theoretical plates was calculated to be 40 for the separation of l-menthol from other reactants.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

THE TEMPORAL PERCEPTION OF MENTHOL1

Although menthol is a common ingredient in pharmaceutical and food products, its sensory properties have not been studied extensively. The objective of this study was to describe and compare the temporal properties of l- and d-menthol. The cooling, burning, and bitterness of two menthol isomers (l-, d-) each at 0.01, 0.02, 0.04, 0.08% (w/v) in aqueous solution were evaluated by 11 trained panelists using time-intensity methodology. The intensity of all three attributes were evaluated continuously from introduction of the sample into the mouth, through expectoration at 10 s, until the termination of the sensation. The l-menthol samples had a greater maximum intensity and longer total duration of cooling and burning sensations than the d-menthol samples. In addition, maximum intensity and total duration of cooling and burn increased with concentration. In contrast, the total duration of the burning sensation was only dependent upon concentration of the l-isomer. Increasing menthol concentration significantly increased maximum intensity and total duration of bitterness for both isomers.     

Intraspecific Variability of the Essential Oil of Ziziphora clinopodioides ssp. rigida from Iran

Hydrodistillated essential oils of Ziziphora clinopodioides ssp. rigida from nine populations of the Lashgardar protected region (Hamedan Province, Iran) were analyzed by using GC and GC/MS techniques to determine the intraspecific chemical variability. Altogether, 39 compounds were identified in the oils, and a relatively high variation in their contents was found. The main constituents of the essential oils were pulegone (0.7–44.5%), 1,8‐cineole (2.1–26.0%), neomenthol (2.5–22.5%), 4‐terpineol (0.0–9.9%), 1‐terpineol (0.0–13.2%), neomenthyl acetate (0.0–7.1%), and piperitenone (0.0–5.4%). For the determination of the chemotypes and the intraspecific chemical variability, the essential oil components were subjected to cluster analysis (CA). The five different chemotypes characterized were Chemotype I (pulegone/neomenthol), Chemotype II (pulegone), Chemotype III (pulegone/1,8‐cineole), Chemotype IV (neomenthol), and Chemotype V (1,8‐cineole/4‐terpineol).     

Metabolism of l-Threonic Acid in Rumex x acutus L. and Pelargonium crispum (L.) L'Hér

l-Threonic acid is a natural constituent in leaves of Pelargonium crispum (L.) L'Hér (lemon geranium) and Rumex x acutus L. (sorrel). In both species, l-[(14)C]threonate is formed after feeding l-[U-(14)C]ascorbic acid to detached leaves. R. acutus leaves labeled with l-[4-(3)H]- or l-[6-(3)H]ascorbic acid produce l-[(3)H]threonate, in the first case internally labeled and in the second case confined to the hydroxymethyl group. These results are consistent with the formation of l-threonate from carbons three through six of l-ascorbic acid. Detached leaves of P. crispum oxidize l-[U-(14)C] threonate to l-[(14)C]tartrate whereas leaves of R. acutus produce negligible tartrate and the bulk of the (14)C appears in (14)CO(2), [(14)C]sucrose, and other products of carbohydrate metabolism. R. acutus leaves that are labeled with l-[U-(14)C]threonate release (14)CO(2) at linear rate until a limiting value of 25% of the total [U-(14)C]threonate is metabolized. A small quantity of [(14)C]glycerate is also produced which suggests a process involving decarboxylation of l-[U-(14)C]threonate.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

Role of eicosanoids, inositol phosphates and extracellular Ca2+ in cell-volume regulation of rat liver

1. In isolated perfused rat liver, the time-course of volume-regulatory K+ efflux following exposure to hypoosmolar perfusate resembled the leukotriene-C4-induced K+ efflux in normotonic perfusion. Omission of Ca2+ from the perfusion fluid had no effect on volume-regulatory K+ efflux, but abolished completely the leukotriene-C4-induced K+ efflux. 2. Volume-regulatory K+ fluxes following hypoosmolar exposure (225 mOsmol l-1) and subsequent reexposure to normotonic media (305 mOsmol l-1) were not significantly affected by the cyclooxygenase inhibitors indomethacin (5 mumol l-1) or ibuprofen (50 mumol l-1), the leukotriene D4/C4-receptor antagonist 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]etha none (YL 171883, 50 microM), the lipoxygenase inhibitor nordihydroguaiaretic acid (20 microM), the phospholipase-A2 inhibitor bromophenacyl bromide (50 microM) or the thromboxane-receptor antagonist 4-[2-(benzenesulfonamido)ethyl]-phenoxyacetic acid (BM 13.177, 20 microM). Also the effects of hypoosmotic cell swelling on lactate, pyruvate and glucose balance across the liver remained largely unaffected in presence of these inhibitors. Neither exposure of perfused rat liver to hypoosmolar (225 mOsmol l-1) nor to hyperosmolar (385 mOsmol l-1) perfusion media affected hepatic prostaglandin-D2 release. 3. When livers were 3H-labeled in vivo by an intraperitoneal injection of myo-[2-3H]inositol about 16 h prior to the perfusion experiment, cell swelling due to lowering the perfusate osmolarity from 305 mOsmol l-1 to 225 mOsmol l-1 led to about a threefold stimulation of [3H]inositol release. The maximum of hypotonicity-induced [3H]inositol release preceded maximal volume-regulatory K+ efflux by about 30 s, but came after the maximum of water shift into the cells. Hypotonicity-induced [3H]inositol release was largely prevented in presence of Li+ (10 mM), but simultaneously inositol monophosphate accumulated inside the liver within 10 min and a small, but significant increase of inositol trisphosphate 1 min after onset of hypoosmolar exposure was detectable. No stimulation of [3H]inositol release was observed during cell shrinkage by switching the perfusate osmolarity from 225 mOsmol l-1 to 305 mOsmol l-1 or from 305 mOsmol l-1 to 385 mOsmol l-1. No stimulation of [3H]inositol release was observed upon swelling of preshrunken livers by lowering the osmolarity from 385 mOsmol l-1 to 305 mOsmol l-1, although the volume-regulatory K+ efflux under these conditions was almost identical to that observed after lowering the osmolarity from 305 mOsmol l-1 to 225 mOsmol l-1. 4.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Generation of Active Oxygen Species Differs in Tobacco and Grapevine Mesophyll Protoplasts

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.     

Tricarboxylic acid-cycle metabolism in brain. Effect of fluoroacetate and fluorocitrate on the labelling of glutamate aspartate, glutamine and γ-amino butyrate

1. The effect of fluoroacetate and fluorocitrate on the compartmentation of the glutamate-glutamine system was studied in brain slices with l-[U-(14)C]glutamate, l-[U-(14)C]aspartate, [1-(14)C]acetate and gamma-amino[1-(14)C]butyrate as precursors and in homogenates of brain tissue with [1-(14)C]acetate. The effect of fluoroacetate was also studied in vivo in mouse brain with [1-(14)C]acetate as precursor. 2. Fluoroacetate and fluorocitrate inhibit the labelling of glutamine from all precursors but affect the labelling of glutamate to a much lesser extent. This effect is not due to inhibition of glutamine synthetase. It is interpreted as being due to selective inhibition of the metabolism of a small pool of glutamate that preferentially labels glutamine.     

Evidence that sabinene is an essential precursor of C(3)-oxygenated thujane monoterpenes

The volatile oil of immature Artemisia absinthium L. leaves contains sabinyl acetate (42%), 3-thujone (32%), sabinene (12%), and α-thujene (3%) as major constitutents, and label from the acyclic precursor [1-³H]geraniol was incorporated, under aerobic conditions, into these thujane-type monoterpenes in proportion to their natural abundance in this tissue. Light had little effect on the synthesis of these monoterpenes from exogenous geraniol; however, at reduced oxygen levels, label from geraniol accumulated in the olefin sabinene while much less sabinyl acetate and 3-thujone were formed, suggesting a route to the ester and ketone by the allylic, nonphotochemical, oxygenation of sabinene. Supporting evidence for the intermediary role of the olefin was provided by isotopic dilution studies in which sabinene, but not α-thujene, blocked formation of the oxygenated derivatives from the labeled precursor. [10-³H]Sabinene was incorporated directly as a substrate in A. absinthium leaves into both [10-³H]sabinyl acetate and 3-[10-³H]thujone. Furthermore, [³H]sabinene was specifically incorporated into 3-thujone in Tanacetum vulgare and into the diastereomeric ketone 3-isothujone in Salvia officinalis, confirming the role of this bicyclic olefin as the essential precursor of C(3)-oxygenated thujane monoterpenes.