Free and ester-bound triterpene alcohols and sterols in cellular subfractions of Calendula officinalis flowers

In the chromoplast fraction and in the chromoplast-free fraction, obtained from Calendula officinalis ligulate flowers, the contents of individual free and ester-bound triterpene alcohols and sterols as well as the fatty acid components of the ester form were determined. It was shown that all sterols and triterpene monols in both forms occur in the two subtractions investigated, whereas all diols are localized only in the chromoplast fraction. The compositions of the fatty acids esterifying monols and sterols were similar to those esterifying diols in the chromoplasts. However, the fatty acids esterifying extra-chromoplast monols and sterols were different. This result indicates that triterpene monol esters are substrates for the biosynthesis of 3-monoesters of diols.     

Distribution of triterpene alcohols in subcellular fractions from Calendula officinalis flowers

The distribution of the triterpene mono- and dihydroxy alcohols was investigated in the subcellular fractions of the flowers of Calendula officinalis. The triterpene monols were found mainly in the chromoplast fraction (68% of total) with smaller amounts in the cell debris, microsomal and supernatant fractions, the mitochondrial fraction was almost devoid of these compounds. Triterpene diols were present exclusively in the chromoplast fraction, 98% in the form of the 3-monoesters and 2% in the form of diesters. It is suggested that the hydroxylation of the triterpene monols to the corresponding diols proceeds in the chromoplasts and the esterified form of the monols is probably the substrate for this reaction.     

The Changes of The Level of Triterpenoids in Calendula officinalis during Vegetation

Using thin-layer chromatography for separation and colorimetric test with CoClj for determination, qualitative and quantitative composition of triterpenoids was estimated in Calendula officinalis dining vegetation. It was found that sterols and oleanolic acid as well as trace amounts of triterpenic monols occur in all organs of the plant during the whole vegetative period. Triterpenic diols and greater quantities of triterpenic monols appear in flowers. The biosynthesis rate of triterpcnoids is highest in young organs of the plant that is in seedlings, young leaves and flower buds. During flowering the content of sterols and oleanolic acid increases in all plant organs except of old leaves. A high level of oleanolic acid in the root during the flowering period implies that oleanolic acid glycosides can be transported from older leaves to underground parts of the plant. Biosynthesis of triterpenic monols, Ψ-taraxasterol and taraxasterol, precedes the formation of the diols faradiol and arnidiol. It can be inferred from the course of accumulation of these compounds that monols are the precursors of diols and that hydroxylation occurs in flowers.     

Stereospecificity of biosynthesis of triterpene alcohols in Calendula officinalis flowers

Flowers of Calendula officinalis were incubated with mevalonic acid doubly labelled with ¹⁴C in position 2 and ³H in positions 2R, 2S, 4R or 5R,S and the [³H/¹⁴C] ratios determined in squalene and pentacylic mono- and dihydroxy-triterpene alcohols and also in some derivatives prepared from the triterpene alcohols. ³H atoms were located in positions 3, 12, 16, 21, 29, 30 of the ursane skeleton, positions 3, 12, 29, 30 of the lupane skeleton and positions 3, 11, 12, 18 of the oleanane skeleton. Stabilization of α- and β-Amyrins, ω-taraxasterol and lupeol occurs with the elimination of a proton from positions 12, 21 and 29 (or 30) respectively. In addition, during hydroxylation of triterpene monols to the corresponding diols a proton is substituted by the hydroxyl group.     

Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-³H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5′-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Biosynthesis of alpinigenine by way of tetrahydroprotoberberine and protopine intermediates

In the biosynthesis of the benzazepine alkaloid alpinigenine a N-methylation step followed by hydroxylation α to nitrogen has now been shown more conclusively to be involved in the transformation of a N-heterocyclic ring system. After feeding Papaver bracteatum plants both the precursors (±)-tetrahydropalmatine-[8,13,14-³H] and (±)-tetrahydropalmatine methiodide-[8,13,14-³H;8-⁴C] an identical mode of abstraction of tritium was observed including a complete loss of the isotope from C-14. The next member in the biogenetic chain, muramine-[8-¹⁴C], was incorporated into alpinigenine very efficiently. Furthermore, using structurally different precursors not utilized for normal alkaloid formation, e.g. 2′-hydroxymethyl-laudanosine-[¹⁴CH₂OH], 13-hydroxymuramine-[8-¹⁴C], the specificity of alkaloid metabolism was examined in the whole plant. Tracer dilution technique was applied to confirm the occurrence in the plant of three established intermediates. Chemical syntheses of four of the alkaloids used during these investigations were developed.     

Subcellular distribution of free and ester-bound triterpene triols in Calendula officinalis flowers

In cellular subfractions obtained from Calendula officinalis ligulate flowers, the contents of individual free and ester-bound triterpene triols, as well as fatty acid components of the ester form, were determined. It was shown that triterpene triols are localized only in the chromoplast fraction, almost exclusively in a free form and in a very small amount as monoesters. The compositions of fatty acids esterifying triols were similar to those esterifying diols in chromoplasts.     

Biosynthesis of artemisia ketone in higher plants

MVA-[2-¹⁴C], IPP-[4-¹⁴C] and DMAPP-[4-¹⁴C] were incorporated (optimum 0.04%–0.8 %) into artemisia ketone by Artemisia annua in a position-specific manner so that the C-5 moiety not containing the carbonyl group was preferentially (87–95 %) labelled. IPP and DMAPP, but not MVA, were similarly utilised in Santolina chamaecyparissus. Feeding of geraniol-[2-¹⁴C] to A.annua resulted in artemisia ketone being labelled in a position indicating extensive degradation of the precursor. ¹⁴C-labelled cis and trans-chrysanthemyl alcohols and chrysanthemates or DMVC were negligibly (< 5 × 10^?4 %) incorporated into artemisia ketone in both species over a range of feeding conditions. (+)-trans-Chrysanthemyl alcohol-[Me¹⁴C] was an effective (ca 2 % incorporation) precursor of the terpenoid part of pyrethrins I and II in flowers of Chrysanthemum cinerariaefolium but ¹⁴C-labelled artemisyl alcohol (3, 3, 6-trimethylheptan-1, 5-dien-4-ol) or (±)-cis-chrysanthemyl alcohol were not detectably incorporated. Although some of the negligible incorporations are probably attributable to compartmentation effects preventing access of precursors to biosynthetic sites, the experiments indicate some limitation of the previously proposed pathways of biogenesis of artemisia ketone and related irregular monoterpenes.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

The biosynthesis of ubiquinone in isolated rat heart cells

Beating heart cells were isolated from the adult rat and the biosynthesis of ubiquinone was studied. These cells were able to incorporate p-hydroxy[U-¹⁴C]benzoate into ubiquinone and some unidentified compounds, presumably intermediates in the biosynthesis of ubiquinone. The unidentified compounds were labile to alkali and were also labeled by [5-³H]-mevalonate and [methyl-³H]methionine, but not by p-hydroxy[carboxy-¹⁴C]benzoate. They appear to be chromatographically different from 5-demethoxy ubiquinone and 5-desmethyl ubiquinone. Addition of unlabeled mevalonate stimulated the incorporation of p-hydroxy [U-¹⁴C]benzoate into ubiquinone and the other compounds. The addition of dimethylsulfoxide to the isolated cells or the isolation medium caused inhibition of ubiquinone biosynthesis. Adriamycin was not inhibitory to the biosynthesis of ubiquinone in the cells. The advantages of these cells are the rapidity and ease in studying the biosynthesis of ubiquinone from various precursors and its regulation.     

Sterol biosynthesis via cycloartenol in Saprolegnia

The Oomycete Saprolegnia ferax incorporates ³H from both cycloartenol-[2-³H] and lanosterol-[2-³H] into its normal sterols cholesterol, fucosterol, desmosterol, and 24-methylenecholesterol. It is concluded that sterol biosynthesis in this organism is via cycloartenol and the taxonomic implications are discussed.     

Translocation of cholesterol from leaves to ripening fruits of Solanum khasianum

After foliar application of [4-¹⁴C]cholesterol to a Solanum khasianum shrub during a 6-week period, cholesterol was recovered not only from untreated leaves, but also from fruits at three different stages of maturity. In addition to free [4-¹⁴C]cholesterol, small amounts of [4-¹⁴C]cholesteryl esters but no [4-C¹⁴]cholesteryl glycosides were found in the fruits, treated, and untreated leaves. Thus, cholesteryl glycosides are probably not involved in the translocation of cholesterol. The implications of cholesterol translocation in the kinetics of solasodine Production are discussed.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

HPLC-based methods for the identification of gibberellin conjugates: Metabolism of [3H]gibberellin A4 in seedlings of Phaseolus coccineus

A series of chromatographic and derivatization techniques has been developed for the identification of radiolabelled gibberellin (GA) conjugates. The methods are based on reversed-phase HPLC, gel permeation chromatography, anion-exchange chromatography, enzymatic hydrolysis and transesterification of conjugates, and derivatization of free GAs to methoxycoumaryl esters. The procedures have been used to identify GA₄-glucosyl ester, GA₄-3-O-glucoside, a GA₃₄-O-glucoside and GA₈-2-O-glucoside, in addition to GA₁ and GA₈, as products of [1,2-³H]GA₄ metabolism in shoots of light-grown Phaseolus coccineus seedlings.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Flavonoids of the flowers of tamarix nilotica

The ethyl ester of kaempferol 3-O-β-D-glucuronide, the methyl and ethyl esters of quercetin 3-O-β-D-glucuronide have been isolated from an aqueous acetone extract of the flowers of Tamarix nilotica. In addition kaempferol 3-O-sulphate-7,4′-dimethyl ether and the free aglycones were isolated. The structures were established by routine methods, by FAB-MS and by ¹³C NMR spectral measurements.