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.     

Interconversion of trans, trans and cis, trans farnesol by enzymes from Andrographis

When trans, trans-farnesol [4,8,12-¹⁴C₃,1-³H₂] is isomerized to cis, trans-farnesol by soluble enzymes from Andrographis paniculata tissue cultures, 50% of the tritium label is lost. The same loss is observed when isomerization occurs in the opposite direction. This is in accordance with the proposed mechanism for isomerization via aldehydes.     

Pathways of carbohydrate oxidation in leaves of Pisum sativum and Triticum aestivum

The aim of this work was to establish the pathways of carbohydrate oxidation used in the dark by leaves of Pisum sativum and Triticum aestivum. Segments of young and mature leaves of pea released the carbons of glucose-[¹⁴C] as ¹⁴CO₂ in the order 3,4 > 1 > 2 > 6 whereas in segments of young and mature leaves of wheat the order was 3,4 > 1 > 6 > 2. The detailed labelling of the constituents of mature leaves of wheat by glucose-[1-¹⁴C], -[2-¹⁴C], -[3,4-¹⁴C], and -[6-¹⁴C] was determined and showed that the high yield of CO₂ from C-6 relative to that from C-2 was due to release of C-6 during pentan synthesis. Estimates were made of the maximum catalytic activities of phosphofructokinase and glucose-6-phosphate dehydrogenase in pea and wheat leaves of three ages. The results of all the above investigations strongly indicate that both pea and wheat leaves in the dark oxidize carbohydrate via glycolysis and the pentose phosphate pathway with the latter accounting for no more than a third of the total. No evidence was obtained of any major change in the relative activities of the two pathways during the development of either type of leaf.     

Sitosterol biosynthesis in Hordeum vulgare

Excised barley embryos cultured on a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly biosynthesized sterols. Two deuterium atoms were present in 24-methylenecycloartanol, 24-methylenelophenol and campesterol and a maximum of four deuterium atoms were incorporated into 24-ethylidenelophenol, stigmasterol and sitosterol. Mevalonic acid-[2-¹⁴C(4R)4-³H₁] was utilized by the barley embryos to give 28-isofucosterol with a ³H-¹⁴C atomic ratio of 3:5 and stigmasterol and sitosterol with a ³H-¹⁴C atomic ratio of 2:5. 24-Methylenelophenol and 24-ethylidenelophenol were isolated from barley seed and 24-ethylidenelophenol-[2,4-³H₃] was incorporated into sitosterol by barley seedlings. These results show that in the production of sitosterol a 24-ethylidenesterol intermediate is produced and it is suggested that this is isomerized to give a Δ^24,(25) sterol prior to reduction to the saturated C₂₉ sterol side chain.     

Ergosterol biosynthesis in Mucor pusillus

Ergosterol, 22-dihydroergosterol, obtusifoliol and 24-methylene-24,25-dihydrolanosterol, isolated from Mucor pusillus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. Ergosterol and 22-dihydroergosterol, isolated from M. pusillus grown in the presence of mevalonic acid-[2-¹⁴C, (4R)-4-³H₁] had ¹⁴C:³H atomic ratios of 5:3. The significance of these results in terms of sterol biosynthesis in this organism in general and alkylation at C-24 in particular is discussed.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Rapid decarboxylation of the products of dark fixation of CO2 in roots of pisum and Plantago

Seedlings of Pisum sativum and excised roots of Plantago major and P. lanceolata were given, in the dark, a pulse of ¹⁴CO₂ in air followed by a chase in ¹²CO₂ in air. A very substantial proportion of the ¹⁴C fixed into organic compounds in the pulse was lost from the tissues in the chase. The activity of NAD malic enzyme in extracts of roots of all three species exceeded their rate of respiration. Azide, 2-n-butylmalonate, and salicylhydroxamic acid each inhibited CO₂ fixation by excised roots of pea. The first two compounds inhibited respiratory gas exchange, but the third stimulated it. Arguments are presented for the widespread diversion of phosphoenolpyruvate from glycolysis to oxaloacetate and thence to malate in the cytosol followed by transport of the malate into the mitochondria for conversion to pyruvate via NAD malic enzyme. No differences, in the above respects, were found between the two species of Plantago.     

Effect of abscisic acid on photosynthetic products of Lemna minor

Lemna minor fronds were grown on nutrient only, or nutrient plus 10^?6M abscisic acid (ABA) for 2 or 8 days. After various ¹⁴CO₂ pulse-chase time periods, the fronds were harvested and the photosynthetic products separated into acidic, lipid, residue, sugar and amino acid fractions. Compared with the control fronds, total ¹⁴C-fixation was 15% higher in the 2 day ABA-treatment and 6% lower in the 8 day ABA-treatment. This pattern was reflected in all the fractions examined, and it appeared that ABA did not alter the distribution of ¹⁴C between the photosynthetic products during the ¹⁴CO₂ pulse. During the chase, less ¹⁴C was lost from the carbohydrate fractions in the ABA-treated fronds than in the control fronds. The results indicate that the previously reported ABA-mediated increase in carbohydrate levels was a consequence of decreased degradation rather than an increase in synthesis from assimilated carbon.     

Proton-transporting urinary epithelia. Reactivity with N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD), a potent inhibitor of the F₀F₁-type H⁺-translocating ATPase, was employed to determine the possible involvement of such an ATPase in urinary acidification. Two methods were used in this approach: (1) the reaction of [¹⁴C]DCCD with tissues involved in urinary acidification and (2) the inhibition of ATPase activity by DCCD. Membrane components from epithelial cells of toad and turtle urinary bladder and brush borders of rabbit kidney were reacted with [¹⁴C]DCCD and analyzed by polyacrylamide gel electrophoresis both before and after extraction with organic solvents. Although a DCCD-binding component was extracted from toad and turtle bladder membranes by chloroform/methanol (2:1, $\text{v}\text{v}$), the binding was not saturable. Analysis of this DCCD-binding component by thin-layer chromatography indicated that there was no ninhydrin reactivity associated with the [¹⁴C]DCCD binding. Moreover, all attempts to precipitate a DCCD-binding protein were unsuccessful. This and other evidence suggested that the observed DCCD binding was to phospholipid. In the second type of experiments, the ATPase activity present in brush borders from rabbit kidney was partially inhibited by DCCD, but at a concentration that is over two orders of magnitude greater than that required for typical DCCD-sensitive ATPase. We conclude from our failure to find positive evidence of a DCCD-reactive protein and from the relative insensitivity of the ATPase to DCCD that either urinary acidification is not accomplished by a typical F₀F₁-type translocating ATPase, or the F₀ has been modified so that the sensitivity to DCCD has been altered or lost.     

Acetate transport by locust rectum in vitro

Everted rectal sacs of Schistocerca gregaria absorb ¹⁴C-acetate from the lumen side at high rates against large electrical and often small concentration differences. Most of the ¹⁴C-activity in the absorbed fluid remains as acetate, but small amounts serve as substrate for aerobic respiration within this tissue. When acetate is substituted for SO₄^?2 or Cl^? in external salines, both short-circuit current (I_sc) and the open-circuit transepithelial potential (PD) increase by as much as 2- to 3-fold. The stimulatory effect of acetate on I_sc and PD exhibits saturation kinetics. The ‘steady-state’ influx of ¹⁴C-acetate from lumen (L) to haemocoel (H) side greatly exceeds efflux (haemocoel to lumen) across short-circuited recta. Over the whole range of acetate concentrations tested, the resulting net flux of acetate is sufficient to explain all of the increase in I_sc caused by this organic anion. Acetate was detected in moderate concentrations in body fluids of locusts. The possible significance of acetate transport in vivo is discussed.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

Urease from leaves of Glycine max and Zea mays

Extracts of Glycine max and Zea mays leaves catalysed the release of ¹⁴CO₂ from [¹⁴C] urea with multiple pH maxima (5.5 and 9.0 for G. max; 5.5, 7.5 and 8.8 for Z. mays). Evidence was obtained that the principal activities, at pH 5.5 and 8.8–9.0 catalysed the same reaction stoichiometry as did urease purified from jackbean seeds (EC 3.5.1.5). The urease activities with these pH optima were not resolved by ammonium sulphate fractionation, DEAE-cellulose chromatography, or gel filtration chromatography. Many structural analogues of urea inhibited leaf urease, the most effective being amino acid hydroxamates, hydroxyurea and selenourea. Allantoic acid and ureidoglycolate are probably not alternative substrates because they showed at most only weak competitive inhibition with respect to radioactive urea.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

On the intracellular site of biosynthesis of α-tocopherol in Hordeum vulgare

When barley seedlings were exposed to ¹⁴CO₂ for 1–3 hr, ¹⁴C was preferentially incorporated into plastoquinone, but only in low amounts i     

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.     

Polyprenyl quinones and α-tocopherol in Calendula officinalis

In various cellular subfractions of Calendula officinalis leaves a study was made of the distribution of polyprenyl quinones and α-tocopherol and the dynamics of their labelling with ¹⁴CO₂ and acetate-[1-¹⁴C] and incorporation of mevalonate-[2-¹⁴C] after 3 hr. It was confirmed that plastoquinone occurs only in the chloroplasts, ubiquinone only in the mitochondria and α-tocopherol in both these subfractions. Phylloquinone was found in the chloroplast and mitochondrial fractions as well as in the post-mitochondrial supernatant. Studies of the dynamics of radioactive precursor incorporation indicated that α-tocopherol is metabolized more rapidly than the polyprenyl quinones studied; the incorporation of mevalonate-[2-¹⁴C] suggests that the side chain of plastoquinone can be synthesized in the cytoplasm and transported to the chloroplasts.     

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.