Metabolism of Monoterpenes : Evidence for the Function of Monoterpene Catabolism in Peppermint (Mentha piperita) Rhizomes

l-Menthone of peppermint leaves is reduced to d-neomenthol which is glucosylated and transported to the rhizome, whereupon the β-d-glucoside is hydrolyzed, the aglycone oxidized back to l-menthone, and this ketone converted to l-3,4-menthone lactone. l-[G-³H]-3,4-Menthone lactone and its labeled progenitors, when incubated with excised mint rhizomes, gave rise to nonvolatile lipids as well as polar metabolites. The lipids thus generated consisted of labeled squalene and phytosterols in the nonsaponifiable fraction and C₁₄-C₂₆ fatty acids in the saponifiable fraction. These results imply degradation of the terpenoid to acetylcoenzyme A and reduced pyridine nucleotide, and reincorporation of label via these products. Starch and soluble carbohydrates were also found to be labeled; however, chemical degradation of the [³H]glucose obtained on hydrolysis of starch indicated the presence of tritium only on interior carbons, suggesting that labeling had occurred via reduced pyridine nucleotides. Analysis of the labeled organic acids revealed the presence of several hydroxy methylacyl intermediates suggesting the operation of a modified β-oxidation pathway in the degradation of the acyclic terpenoid skeleton. The results indicate that monoterpenes transported to the rhizome are oxidized to yield acetyl-coenzyme A and reduced pyridine nucleotides, and suggest that metabolic turnover of monoterpenes in mint represents a mechanism for recycling carbon and energy from foliar terpenes into other metabolites of the rhizome.     

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.     

Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis)

The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the β-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-³H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess β-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-¹⁴C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C₁₄ to C₂₀) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.     

Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes

Abstact  A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium- and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-⁵H-dibenzo[a,d]cyclopenten-5,1-imine ([G-³H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-³H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci/mol, respectively. The isotopomeric distribution of deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal ion injection. Mean deuterium incorporation per ligand molecule was 11.09 and 3.21 atoms for [G-³H]MK-801 and [G-³H]-7-OH-DPAT, respectively. The isotope label was shown to be distributed all over the ligand molecule. The radioreceptor binding of tritium-labeled ligands [G-³H]MK-801 and [G-³H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-³H]MK-801 specifically binds to hippocampus membranes with K _d 8.3 ± 1.4 nM, B _max being 3345 ± 300 fmol/mg protein. The [G-³H]-7-OH-DPAT ligand specifically binds to rat striatum membranes with K _d 10.01 ± 0.91 nM and B _max 125 ± 4.5 fmol/mg protein. It was concluded that the HSCIE reaction can be used for the preparation of highly tritium-labeled (+)-MK-801 and 7-OH-DPAT with retention of their physiological activities. Original Russian Text ? Yu.A. Zolotarev, Yu.Yu. Firsova, A. Abaimov, A.K. Dadayan, V.S. Kosik, A. V. Novikov, N.V. Krasnov, B. V. Vaskovskii, I.V. Nazimov, G.I. Kovalev, N.F. Myasoedov, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 3, pp. 323–333.     

Metabolism of Monoterpenes : EVIDENCE FOR COMPARTMENTATION OF l-MENTHONE METABOLISM IN PEPPERMINT (MENTHA PIPERITA) LEAVES

Previous studies have shown that the monoterpene ketone l-[G-(3)H]-menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaf discs 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-beta-d-glucoside (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). The metabolic disposition of the epimeric reduction products of the ketone, which is a major constituent of peppermint oil, is highly specific, in that little neomenthyl acetate and little menthyl glucoside are formed. However, when l-[3-(3)H]menthol and d-[3-(3)H]neomenthol are separately administered to leaf discs, both menthyl and neomenthyl acetates and menthyl and neomenthyl glucosides are formed with nearly equal facility, suggesting that the metabolic specificity observed with the ketone precursor was not a function of the specificity of the transglucosylase or transacetylase but rather a result of compartmentation of each stereospecific dehydrogenase with the appropriate transferase. A UDP-glucose:monoterpenol glucosyltransferse, which utilized d-neomenthol or l-menthol as glucose acceptor, was demonstrated in the 105,000g supernatant of a peppermint leaf homogenate, and the enzyme was partially purified and characterized. Co-purification of the acceptor-mediated activities, and differential activation and inhibition studies, provided strong evidence that the same UDP-glucose-dependent enzyme could transfer glucose to either l-menthol or d-neomenthol. Determination of K(m) and V for the epimeric monoterpenols provided nearly identical values. The acetylcoenzyme A:monoterpenol acetyltransferase previously isolated from peppermint extracts (Croteau, Hooper 1978 Plant Physiol 61: 737-742) was re-examined using l-[3-(3)H]menthol and d-[3-(3)H]neomenthol as acetyl acceptors, and the K(m) and V for both epimers were, again, very similar. These results demonstrate that the specific in vivo conversion of l-menthone to l-menthyl acetate and d-neomenthyl-beta-d-glucoside cannot be attributed to the selectivity of the transferases, and they clearly indicate that the metabolic specificity observed is a result of compartmentation effects.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-³H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-¹⁴C]inositol, myo-[2-³H]inositol, l-[5-¹⁴C]arabinose, and d-[5R,5S-³H]xylose.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Conversion of [1-3H2,G-14C]geranyl pyrophosphate to cyclic monoterpenes without loss of tritium

Soluble enzyme preparations from Salvia officinalis convert the acyclic precursor [1-³H₂,G-¹⁴C]geranyl pyrophosphate to cyclic monoterpenes of the pinane (α-pinene,β-pinene), isocamphane (camphene), p-menthane (limonene,1,8-cineole), and bornane (bornyl pyrophosphate, determined as borneol) type without loss of tritium, and without significant conversion to other free acyclic intermediates. Similarly, [1-³H₂,G-¹⁴C]geraniol is converted in intact S. officinalis leaves to the cyclic monoterpene olefins and 1,8-cineole, as well as to isothujone and camphor, without loss of tritium from C(1). These results clearly eliminate transcis isomerization of geranyl pyrophosphate to neryl pyrophosphate via aldehyde intermediates prior to cyclization, and they support a scheme whereby the trans precursor is cyclized directly by way of a bound linaloyl intermediate.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

Taraxacoside,a type of acylated γ-butyrolactone glycoside from taraxacum officinale

In addition to the four new sesquiterpene lactones previously identified, a new acylated γ-butyrolactone glucoside, taraxacoside, was isolated from the roots of Taraxacum officinale. Its structure was elucidated mainly by ¹H and ¹³C NMR studies as β-O-[4-O-(p-hydroxyphenylacetyl]β-D-glucopyranosyl]-β-hydroxy-γ-butyrolactone. This seems to be the first instance of the detection of a monocyclic five-membered, saturated lactone O-glycoside. Additionally, p-hydroxyphenylacetic acid was identified for the first time as an acylating acid in a sugar ester.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6-C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Hér

Conversion of d-[5-³H,6-¹⁴C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a ³H:¹⁴C ratio of 4 to be compared with 9, the ratio in d-[5-³H,6-¹⁴C]glucose initially. Conversion of d-[6-³H,6-¹⁴C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a ³H:¹⁴C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.     

PREFERENTIAL LABELING OF RAT LYMPHOCYTES WITH A RAPID RATE OF TURNOVER BY TRITIATED DEOXYCYTIDINE

Lymphocytes in thymic cortex and germinal centers of lymphoid tissues are labeled intensely with generally labeled tritiated deoxycytidine [G-³H]dCyd whereas they are weakly labeled with methyl tritiated deoxythymidine [methyl-³H]dThd of the same specific activity, not only by single injection but also by an intensive injection schedule. [G-³H]dCyd can be used to label short-lived lymphocytes strongly, although not specifically. The distribution patterns of labeled lymphocytes were different depending on the injection schedules of [G-³H]dCyd. [G-³H]dCyd can be used as a precursor molecule for cytosine and also thymine found in DNA. The ratios of radioactive thymine to cytosine measured biochemically on DNA extracted from radioactive lymphocytes labeled by the various schedules indicate strongly that short- and long-lived lymphocyte populations have different abilities to utilize pyrimidine nucleosides for DNA synthesis.     

d-Aspartate binding sites in rat Harderian gland

Radioligand binding of d-[³H]aspartic and l-[³H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K _d = 860 ± 28 nM, B _max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K _d = 580 ± 15 nM and B _max = 51.3 ± 0.8 pmol/mg protein). l-[³H]glutamate had higher affinity and a greater percentage of specific binding than did d-[³H]aspartate. The pharmacological binding specificity of l-[³H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[³H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.     

Evidence of the existence of two different intraneuronal pools from which pharmacological agents can release serotonin

The effect of various drugs on the release of [³H]-serotonin from synaptosomes of reserpine-treated rats was compared with that obtained with synaptosomes of untreated animals.The increase in [³H]-serotonin release induced by d-fenfluramine was virtually abolished by reserpine; the effect of d-norfenfluramine, the main metabolite of fenfluramine, was instead enhanced in synaptosomes of reserpine treated animals. [³H]-serotonin release induced by l-isomers of fenfluramine or norfenfluramine was increased or not affected, respectively, after reserpine treatment.The effects of other drugs, known to activate serotonin mechanisms such as metachlorophenylpiperazine and quipazine, like d-norfenfluramine, were increased by the reserpine treatment.The present data show that [³H]-serotonin can be released by drugs from two pools with different sensitivity to reserpine. The reserpinized synaptosomes could provide useful information on the mechanisms of action of drugs acting on brain serotonin.     

Effect of lithium and sodium ions on opiate-and dopamine-receptor binding

The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-³H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [³H]naloxone and [³H]DHM. Binding of [³H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [³H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.     

7-Amino-actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA

Fluorescence, CD, absorption, and ¹H-nmr studies are reported for complexes of 7-amino-actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG-dC)· poly(dG-dC). The optical spectra for the 7-amino-actinomycin D complex with pdG-dC, pdG-dC-dG-dC and pdC-dG-dC-dG are similar in shape to the 7-amino-actinomycin D complex with either DNA or poly(dG-dC). The changes in the ¹H chemical shifts of the 7-amino-actinomycin D and the pdG-dC resonances that accompany complex formation show that 7-amino-actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The magnitudes of the induced chemical shifts for the 7-amino-actinomycin D complex formation with pdG-dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The pdN-dG dinucleotides (N = C, A, or T) form stacked complexes with 7-amino-actinomycin D. The presence of the 7-amino-group results in a larger dimerization constant (in aqueous solution) for 7-amino-actinomycin D [K_D(6°C) = 4.4 × 10³M^?1], as compared to actinomycin D [K_D(6°C) = 1.7 × 10³M^?1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7-amino-actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG-dC)·poly(dG-dC), and poly(dA-dC)·poly(dG-dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans

The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations (e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [³H]leucine, [³H]thymidine, or [³H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [¹⁴C]lactose and [¹⁴C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [¹⁴C]α-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20°C results in the leakage of accumulated [¹⁴C]α-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [¹⁴C]glycine and of [¹⁴C]α-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate-dependent transport of [¹⁴C]glycine but not vectorial phosphorylation-mediated transport of [¹⁴C]α-methyl glucoside. Thus, capsidiol's mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.