Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly ¹⁴C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [carboxy-¹⁴C]-, and [Me-¹⁴C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.     

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.     

An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis

A sensitive radioassay is described for the overall biosynthetic activity of the multienzymatic protein which catalyzes the first three reactions of de novo pyrimidine biosynthesis in mammals. The ability of the multienzymatic protein to synthesize dihydroorotate can be assayed using $\text{[}{}^{14}\text{C]HCO}{}_3{}^{\mathrm{?}}$, l-[¹⁴C]aspartate, or [${}^{14}\text{C}$] carbamyl phosphate as substrate. The synthesis of the final product, l-dihydroorotate, may be coupled to synthesis of orotidine 5′-monophosphate to overcome the unfavorable equilibrium existing between l-dihydroorotate and its precursor, N-carbamyl-l-aspartate, in the physiological pH range (Christopherson, R. I., and Jones, M. E., 1979, J. Biol. Chem.254, 12506–12512). l-Aspartate and all pyrimidine intermediates from carbamyl phosphate to orotidine 5′-monophosphate can be clearly separated by ion-exchange chromatography in a single dimension on polyethyleneimine-cellulose chromatograms and carbamyl phosphate and its degradation product cyanate may be quantitated directly along with the other intermediates.     

The function of the pentose phosphate pathway in Phaseolus mungo hypocotyls

The metabolism of d-gluconate-[1-¹⁴C] and -[6-¹⁴C] by segments from etiolated hypocotyls of Phaseolus mungo has been studied. The release of ¹⁴CO₂ from gluconate-[1-¹⁴C] was greater than that from gluconate-[6-¹⁴C] in all parts of hypocotyls examined. Incorporation of the radioactivity from gluconate-[6-¹⁴C] into RNA, lignin and aromatic amino acid fractions was greater in the upper (younger) part of the hypocotyls. Incorporation into sugars was greater in the lower (more mature) parts.     

Chloroethene Biodegradation in Sediments at 4°C

Microbial reductive dechlorination of [1,2-¹⁴C]trichloroethene to [¹⁴C]cis-dichloroethene and [¹⁴C]vinyl chloride was observed at 4°C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-¹⁴C]cis-dichloroethene and [1,2-¹⁴C]vinyl chloride to ¹⁴CO₂ also was observed under these conditions.     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.     

Titles of related papers in other sections

Culture of Trypanosoma cruzi (Tulahuen strain) in the presence of ethidium bromide (1–20 μg/ml) resulted in dyskinetoplasty and inhibition of growth, to an extent depending on the dye concentration and the medium composition. The ethidium bromide-induced dyskinetoplasty caused a decrease of (a) the cytochrome content of epimastigotes (a,a₃ and b species); (b) the rate of respiration (endogenous or supported by D-glucose); and (c) the rate of production of ¹⁴CO₂ from [2-¹⁴C]acetate and [1-¹⁴C]glucose. [2-¹⁴C]Acetate oxidation to ¹⁴CO₂ was affected by dyskinetoplasty more than [1-¹⁴C]glucose oxidation, particularly at the exponential growth phase. With dyskinetoplastic epimastigotes, diminution of ¹⁴CO₂ production from [2-¹⁴C]acetate largely exceeded that of oxygen uptake, while with [1-¹⁴C]glucose, ¹⁴CO₂production and respiration were affected to about the same extent. Dyskinetoplasty also decreased the incorporation of [2-¹⁴C]acetate carbon into intermediates of the tricarboxylic acid cycle and related amino acids, and modified the distribution pattern of ¹⁴C in accordance with the decrease of respiration. Reduction of cytochrome content of epimastigotes by restriction of heme compounds during growth decreased ¹⁴CO₂ production from [2-¹⁴C]acetate, like the ethidium-induced dyskinetoplasty. The same occurred after inhibition of electron transfer by antimycin and cyanide, though to a much more significant extent, thus confirming the functional association of electron transport at the mitochondrial cytochrome system of T. cruzi and the enzymatic reactions of the tricarboxylic acid cycle.     

Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-¹⁴C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.     

Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are 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.     

In vitro substrate utilization for lipid synthesis in liver explants from hyperthyroid chickens

• 1.1. Indian River male broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 18, 24 and 30% protein + 0 or 1 mg triiodothyronine (T₃)/kg of diet to study energetic costs of lipogenesis and the use of various substrates for in vitro lipogenesis.
• 2.2. De novo lipid and CO₂ production were determined in the presence of [1-¹⁴C]pyruvate, [2-¹⁴q]pyruvate, [3-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine.
• 3.3. Oxygen consumption was determined in mitochondrial preparations to estimate the energetic costs in expiants synthesizing lipid.
• 4.4. Radiolabeled CO₂ derived from [1-¹⁴C]pyruvate was used as an estimate of coenzyme A availability in liver expiants. Lipids derived from [2-¹⁴C]pyruvate, [2-¹⁴C]acetate and [U-¹⁴C]alanine estimate relative substrate efficiency.
• 5.5. Labeled CO₂ production from [1-¹⁴C]pyruvate was greatest in that group fed a 12% protein diet and least in the group fed a 30% protein diet.
• 6.6. In addition, T₃ increased CO₂ production from [1-¹⁴C]pyruvate.
• 7.7. The production of ¹⁴CO₂ from the second carbon of pyruvate or acetate was increased by T₃.
• 8.8. The low-protein diet (12% protein) increased (P <0.05) lipogenesis.
• 9.9. Adding T₃ to the diets decreased carbon flux into lipid from all substrates, but increased CO₂ production from all substrates without changing stage 3 and 4 respiration rates in mitochondrial preparations.
• 10.10. These observations imply that coenzyme A availability may have regulated de novo lipogenesis in the present study.
• 11.11. It was also concluded that previously noted effects of T₃ on intermediary metabolism may involve metabolic pathways that do not involve changes in mitochondrial function.

     

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.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.     

Long Chain (C(20) and C(22)) Fatty Acid Biosynthesis in Developing Seeds of Tropaeolum majus: AN IN VIVO STUDY

The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C₂₀ and C₂₂ fatty acids, developing seed tissue was incubated with various ¹⁴C-labeled precursors. Incubation with [1-¹⁴C]acetate produced primarily cis-11-[1-¹⁴C]eicosenoate and cis-13-[1,3-¹⁴C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-¹⁴C]oleate also formed from [¹⁴C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24-hour period. Under suitable conditions, nasturtium seed could also produce [¹⁴C]stearate, [¹⁴C]eicosenoate, and [¹⁴C]docosenoate from [1-¹⁴C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently.     

Biosynthesis of lipids in chloroplasts isolated from jack pine needles

Suspensions of isolated pine needle chloroplasts were shown to incorporate galactose from UDP galactose-[¹⁴C] into galactolipids. The incorporation of the label among galactolipids was always considerably higher in the monogalactosyl diglycerides than in the digalactosyl diglycerides. The galactosyl incorporation into both galactolipid fractions was optimal at pH 8.0 and was inhibited by sulphydryl reagents (p-chloromercuribenzoate, N-ethyl maleimide and CdCl₂). The chloroplast preparations were also able to biosynthesize various phospholipids and galactolipids from palmitoyl-[1-¹⁴C]-CoA; the major portion of the label appeared in phosphatidyl choline. The incorporation of palmitic-[1-¹⁴C] acid into various lipids was very poor compared to that of palmitoyl-[1-¹⁴C]-CoA. However, addition of ATP and CoA markedly stimulated lipid biosynthesis from palmitic-[1-¹⁴C] acid, suggesting the presence of activating enzymes. These chloroplast suspensions did not show any de novo fatty acid synthesis.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Hemolymph volume of noninfected and Plasmodium berghei-infected Anopheles stephensi.

The hemolymph volume of Anopheles stephensi adult female mosquitoes was determined by a radioisotope dilution technique. [carboxy-¹⁴C]Inulin was injected into the hemocoels of mosquitoes with a calibrated capillary needle. After sufficient time for thorough mixing, the labeled hemolymph was collected from groups of 50 mosquitoes by a centrifugation technique. Total hemolymph volume was calculated by a conventional formula for radioisotope dilution. The mean hemolymph volume of the newly emerged adult female mosquitoes was 336 nl/mosquito. The ratio of hemolymph volume to body weight was 0.25 μl/mg body wt. By 14 days after emergence, hemolymph volume had dropped to 190 nl/mosquito. Infection of mosquitoes with the rodent malaria parasite, Plasmodium berghei, had no significant effect on hemolymph volume of the mosquito.