Biosynthesis of triterpenoid sapogenols in soybean and alfalfa seedlings

By incubation of germinating soybeans with mevalonate-[2-¹⁴C] (MVA), radioactivity was incorporated into four sapogenols which were identified by TLC. Unequivocal evidence for the identity of three of the four sapogenols was provided by co-crystallization to constant specific radioactivity. The partition of incorporated radioactivity into lipid- and water-soluble fractions and the pattern of radioactivity of individual sapogenols varied with the mode of administering labeled substrates to soybean seedlings, such as incubation of germinating soybeans with MVA-[2-¹⁴C], immersion of roots into MVA-[2-¹⁴C] or foliar application of squalene-[¹⁴C]. When alfalfa seedlings were incubated with MVA-[2-¹⁴C], about two-thirds of the radioactivity incorporated into the sapogenols was associated with medicagenic acid.     

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.     

Biosynthesis of monoterpenes in plants from 14C-labelled acetate and CO2

Degradation of (+)-isothujone biosynthesized by Tanacetum vulgare or Thuja plicata from acetate-[1-¹⁴C], -[2-¹⁴C] and -[2-³H₃] or from CO₂-[14C] at physiological concentration revealed a pattern of asymmetric labelling whereby tracer predominantly (72–98% resided in that part of the skeleton derived from IPP. This is similar to the patterns previously obtained for uptake of MVA-[2-¹⁴C] but differed from those reported in other species with acetate-[¹⁴C] as precursor. Within the IPP-derived moiety the 3 parts derived from acetate units were not equivalently labelled. Partial degradations of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium after uptake of ¹⁴C-labelled acetate or CO₂ showed that the C-2 units of the skeletons of these monoterpenes were also labelled to widely differing extents and these patterns persisted over a range of feeding and seasonal conditions. These results suggest that metabolic pools of acetyl-CoA and/or acetoacetyl-CoA exist in these plants. The general occurrence of such pools and the consequent nonequivalent labelling patterns in secondary metabolism could invalidate biosynthetic conclusions drawn from partial degradations of labelled natural products.     

Biosynthesis of (+)-car-3-ene in Pinus species

Degradation of (+)-car-3-ene biosynthesized from MVA-[2-¹⁴C] in Pinus palustris or Pinus sylvestris proved that the C-4 atom of the monoterpene is derived from C-2 of MVA rather than C-4 as has been hitherto assumed. The pro-2S hydrogen of MVA is stereospecifically lost in the formation of the Δ³-double bond. These results delineate possible routes for the biosynthesis of the carane skeleton.     

Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare

Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-¹⁴C] and MVA-[2-¹⁴C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[¹⁴C] and terpinen-4-ol-[¹⁴C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-¹⁴C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.     

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.     

Biosynthesis of artemisinin in Artemisia annua

The isotope ratios (³H:¹⁴C) in arteannuin B and artemisinin biosynthesized in Artemisia annua from [4R-³H₁,2-¹⁴C]-, [5-³H₂,2-¹⁴C]- and [2-³H₂,2-¹⁴C](3RS)- mevalonate have revealed that two specific 1,2-hydride shifts take place during the oxidation and lactonization of the germacrane skeleton to yield dihydrocostunolide. The gem-methyls of DMAPP retain their identity until the final steps of artemisinin biosynthesis. Arteannuin B is considered to be a late precursor of artemisinin and the following biosynthetic sequence is suggested: farnesylpyrophosphate → germacrane skeleton → dihydrocostunolide → cadinanolide → arteannuin B → artemisinin.     

Early stages of ecdysteroid biosynthesis: The role of 7-dehydrocholesterol

The synthesis of [4-¹⁴C]cholesta-4,6-dien-3-one and [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one is described. Both [4-¹⁴C]cholest-4-en-3-one and [4-¹⁴C]cholesta-4,6-dien-3-one were not incorporated significantly into ecdysteroids compared to [1α,2α-³H]cholesterol in fifth instar and maturing adult female Schistocerca gregaria. Similarly, [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one was not incorporated significantly in the latter system. The results suggest that none of the three ¹⁴C-substrates are intermediates in ecdysteroid biosynthesis from cholesterol, although possible complications from permeability barriers cannot be discounted. [4-¹⁴C, 7-³H]7-dehydrocholesterol has been synthesized and incorporated into ecdysteroids in adult female Schistocerca gregaria and in Spodoptera littoralis pupae. Although approximately half the tritium was eliminated during ecdysteroid synthesis in S. gregaria, there was essentially complete retention of the tritium in Spodoptera. The results support the direct incorporation of 7-dehydrocholesterol into ecdysteroids and not via cholesterol. A possible explanation for the loss of appreciable tritium in S. gregaria is discussed.     

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 tropic acid: Feeding experiments with cinnamoyltropine and littorine

The administration of cinnamoyl-[2-¹⁴C]-tropine-[N-methyl-¹⁴C] to Datura stramonium plants resulted in the formation of labeled atropine and scopolamine. However the atropine was found to have almost all its radioactivity located on the N-methyl group of the alkaloid, indicating that the administered ester had undergone hydrolysis in the plant affording tropine and cinnamic acid, the latter not being utilized for the biosynthesis of tropic acid. Dual labeled RS-littorine (3β-(2-hydroxy-3-phenylpropionyloxy-[1-¹⁴C]-tropane-[3β-³H]) was also fed to D. stramonium and radioactive atropine was obtained. However the drastic change in the ³H:¹⁴C ratio found in the atropine indicated that the littorine was not converted directly to the alkaloid, and it is suggested that the littorine is hydrolysed _{in vivo} to tropine and phenyl-lactic acid, the latter undergoing rearrangement to tropic acid prior to esterification with tropine.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

Biosynthesis of the phytoalexin wyerone in Vicia faba

In contrast to earlier results [1-¹⁴C] acetate, [2-¹⁴C] malonate and [n 9,10-³H] oleate show significant incorporations into wyerone and related Vicia faba phytoalexins, following infection by Botrytis cinerea.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino) ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-³H]taurine and [1-¹⁴C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of ³H and ¹⁴C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Measuring plasma fatty acid oxidation with intravenous bolus injection of 3H- and 14C-fatty acid

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-¹⁴C]palmitate versus [9,10-³H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (¹⁴C- or ³H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [³H]acetate recovery as ³H₂O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-¹⁴C]acetate recovery as expired ¹⁴CO₂ was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-¹⁴C]palmitate and [9,10-³H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [³H]palmitate. In conclusion, intravenous boluses of [9,10-³H]palmitate versus [1-¹⁴C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

Biosynthesis of Camptotheca acuminata alkaloids

Tracer feeding experiments with Camptotheca acuminata plants show that [1′-¹⁴C]L-tryptophan, [Ar-³H₄]L-tryptophan, [Ar-³H₄,1′-¹⁴C]tryptophan, [1′-¹⁴C]-tryptamine, [2-¹⁴C]DL-mevalonate, and [2-¹⁴C]geraniol-[2-¹⁴C]nerol are incorporated into camptothecin. Direct stem injection of the labeled precursors into C. acuminata plants resulted in a substantial increase in the activity of isolated Camptotheca alkaloids as compared to root feeding of the same tracer.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: The role of an isoflav-3-ene

Feeding experiments in CUCl₂- and UV-treated lucerne (Medicago sativa) seedlings have shown that demethylhomopterocarpin- [6a-³H, Me- ¹⁴C] is incorporated into vestitol and sativan without any loss of ³H label, and vestitol-[3-³H, Me- ¹⁴C] is similarly incorporated into demethylhomopterocarpin and sativan with retention of the ³H: ¹⁴C ratio. Thus, an isoflav-3-ene intermediate in the interconversion of demethylhomopterocarpin and vestitol is excluded. 7,2′-Dihydroxy-4′-methoxyisoflav-3-ene- [Me-¹⁴C] was not incorporated into the three phytoalexins, but was an excellent precursor of 9-O-methylcoumestrol, as also was 7,2′-dihydroxy-4′-methoxyisoflav-3-en-2-one- [Me-¹⁴C]. A biosynthetic pathway to coumestans via isoflav-3-enes and 3-arylcoumarins is proposed. A metabolic scheme in M. sativa interrelating eight classes of naturally occurring isoflavonoids is presented.     

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.     

Identification of 3-hydroxy-6-methyl-2H-pyran-2-one from pyrolysis of phosphoric acid-treated cellulosic materials

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5-³H]glucose 6-phosphate into myo-[2-³H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the ³H/¹⁴C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5-³H, 1-¹⁴-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of ³H when UDP-d-[5-³H, 1-¹⁴C]glucose is oxidized by UDP-d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.