Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans

The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.     

Precursors of ricinine in the castor bean plant

Isotopic tracer experiments confirmed that glycerol and succinic acid are good precursors of the pyridine ring of ricinine in castor bean plants. Tritium from C-2 was lost from tritiated glycerol while tritium from C-1 was retained. Thus a derivative of dihydroxyacetone is likely to be intermediate. By simultaneous feeding of glycerol-1-(3)-[³H] and succinic acid-2(3)-[¹⁴C], it was hoped to find precursors of ricinine containing both labels, but none could be found. There was no evidence for the appearance of labeled quinolinic acid, which is presumed to be a precursor of ricinine.     

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [³H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinic acid riboside → nicotinic acid mononucleotide → nicotinic acid adenine dinucleotide → NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-¹⁴C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-¹⁴C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.     

Pyridine nucleotide biosynthesis in Claviceps

Claviceps purpurea grown on synthetic medium incorporated labeled [7-¹⁴]nicotinic acid and [7-¹⁴C]nicotinamide into NaMN, des-NAD, NAD, and NADP. Label also appeared in NMN and N-methyl nicotinamide. The specific activities of NAD, NADP, and NMN are compatible with the operation of the Preiss-Handler pathway of NAD biosynthesis (nicotinic acid → NaMN → des-NAD → NAD → NADP). The relative amounts of NaMN:des-NAD:NAD and NADP were about 8:1:36:10 on incubation of Claviceps with nicotinic acid for 6 hr. The incorporation of nicotinamide into NAD proceeds mainly by conversion to nicotinic acid catalyzed by nicotinamide deamidase.Tryptophan ([U-¹⁴C]benzene ring) was incorporated into NAD demonstrating the presence of the tryptophan-nicotinic acid pathway. No qualitative difference in pyridine nucleotide intermediates was noted in C. purpurea CPM, which does not produce clavine alkaloids, and Claviceps 47A which does produce clavine alkaloids.     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.     

Thin-layer chromatographic separation of intermediates of the pyridine nucleotide cycle

A rapid thin-layer chromatographic procedure for separation of the compounds comprising the intermediates in the salvage pathway known as the pyridine nucleotide cycle plus quinolinic acid and the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate is described. The method utilizes silica gel high-performance thin-layer plates and a mobile phase of methanol, tetrabutylammonium hydroxide, and acetonitrile. The time required for analysis is greatly reduced and results in greater than 96% purity of each migrating compound.     

Protein and RNA synthesis in the pea aphid,Acyrthosiphon pisum during different stages of development

Protein and RNA synthesis in the pea aphid, Acyrthosiphon pisum were studied at specific intervals during the entire life cycle. The pool size of [¹⁴C]-l-leucine in the aphid free amino acid pool and the incorporation of the same into the protein fraction increased with growth and reached a maximum between 12 and 16 days, corresponding to the period of active reproduction. The pool size of [³H]-orotic acid in the nucleotide pool and the incorporation of the same into the RNA fraction of the pea aphid also showed similar patterns.     

Translocation and Metabolism of Ricinine in the Castor Bean Plant, Ricinus communis L

Ricinine-3,5-¹⁴C (N-methyl-3-cyano-4-methoxy-2-pyridone) administered to senescent leaves of Ricinus communis L. was translocated to all other tissues of the plant. Developing fruit and especially seeds were found to be labeled the most rapidly. Young growing leaves and other developing tissues of the plant imported ricinine from the senescent leaves much more quickly than mature leaves. Relative intensities of the radioactive ricinine imported and deposited in various tissues indicate a possible functional role of ricinine in the castor bean plant. Data on N-demethyl ricinine presented here may stimulate interest in the possible physiological role of the ricinine to N-demethyl ricinine interconversion.     

Studies on the biochemistry and fine structure of silica-shell formation in diatoms

Summary The distribution of radioactivity in ethanol-water-soluble compounds after short periods of photosynthetic incorporation of ¹⁴CO₂ is consistent with the operation of the photosynthetic carbon reduction (PCR) cycle in the fresh water diatom Navicula pelliculosa. Incorporation of ¹⁴CO₂ for extended time periods established the presence of the intermediates of the PCR and tricarboxylic acid (TCA) cycles, amino acids, and organic acids; free sugars were not observed. The main labelled soluble carbohydrate was a glucan. Hydrolysis of the radioactive insoluble material indicated the presence of carbohydrates containing several distinct sugars, and proteins with the usual amino-acid composition. During silicon starvation of exponentially growing cultures, rates of incorporation of both ³²P _i and ¹⁴CO₂ decreased. Incorporation into the lipid increased, with a corresponding decrease into protein and carbohydrate. Reintroduction of Si to staryed cells led to an increased rate of incorporation of both isotopes, and transient changes in the radioactivity in most metabolic intermediates investigated. After 30 min the radioactivity in all PCR cycle intermediates, except phosphoglyceric acid, had increased by about 300%. The radioactivity of citrate and -keto-glutarate increased, whereas that of other TCA-cycle intermediates decreased. An initial decrease in the levels of glutamate, aspartate and glutamine was apparently reversed by cleavage of glutamate-aspartate peptides, as radioactivity of other amino acids increased. Incorporation into the soluble glucan and into protein increased markedly although the rate of incorporation into insoluble carbohydrates remained constant.     

Carbon Assimilation in Photoheterotrophic Cells of Peanut (Arachis hypogaea L.) Grown in Still Nutrient Medium

The relative transport of photosynthetic and dark carboxylation products in photoheterotrophic cells of Arachis hypogaea L. var. TMV-3 at varied phases of growth were determined. Despite the presence of an equally competent photosynthetic apparatus as determined from ¹⁴CO₂ incorporation rates in the dark and light, pulse-chase experiments revealed little or no change in the radioactivity of the C₃ intermediates but rapid disappearance of label from the dark carbon assimilates (malate and other tricarboxylic acid cycle intermediates) with a simultaneous increase in the aminoacid pool in early log-phase (10 days old) cells. However, significant flow of carbon through the photosynthetic intermediates resulting in the accumulation of sugars occurred in the late log-phase (34 days old) cells. Limitation of exogenous sugar in the nutrient milieu and depletion of reserve carbohydrates stored in starch of the chloroplasts of the cells were considered as the decisive factors in promoting transport of C₃ cycle intermediates through the reductive pentose phosphate pathway in photoheterotrophic cells. The observed drain of radioactivity even from the small amounts of tricarboxylic acid cycle intermediates synthesized during photosynthesis into glutamate indicated that the transport of carbon through the nonautotrophic pathway is not controlled by these factors.     

Biosynthesis of pyridine alkaloids from Tripterygium wilfordii

Nicotinic acid-6-¹⁴C and nicotinamide adenine dinucleotide-carbonyl-¹⁴C were rapidly metabolized in T. wilfordii Hook. with formation of all compounds in the pyridine nucleotide cycle. Nicotinic acid-6-¹⁴C and the nicotinamide moiety of NAD were efficiently incorporated into wilfordic acid and hydroxywilfordic acid, the pyridinium moieties of the ester alkaloids. The structures of wilfordic acid and hydroxywilfordic acid were confirmed using GLC-MS. The molecular formulae of the four isolated alkaloids were determined by high resolution MS and agreed with earlier results based on elemental analysis.     

Pathway of fructosan synthesis in leaf bases of Dactylis glomerata

Chemical analysis of leaf base tissue of Dactylis glomerate failed to detect any low MW oligosaccharide intermediates during fructosan synthesis. Extracts of tissue harvested at various times after the incorporation of ¹⁴CO₂ showed a decline in radioactivity in sucrose and an equivalent rise in high MW fructosan with no significant accumulation of radioactivity in oligosaccharides. No evidence was obtained for the existence of nucleotide fructose in the tissue, indicating that fructosan synthesis occurs by direct transfer of fructosyl residues from sucrose to the polymer.     

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.     

Synthesis of small polynucleotide chains in thymine-depleted bacteria

Bacteria that are depleted of intracellular thymidine nucleotide pools incorporate [³H]thymine at full specific activity, allowing the detection of early intermediates in DNA replication. A short pulse of [³H]thymine is incorporated almost exclusively into very small DNA chains which, during further incorporation of thymine, are converted into larger chains and high molecular weight DNA. The synthesis of these small DNA chains depends on the products of dna genes B, E and G. Analysis of the DNA by gel filtration on Sepharose 2B revealed an abundance of extremely short DNA chains while the frequency of larger chains decreased exponentially with increasing size. This size distribution of small DNA chains suggests a mechanism of DNA replication in which larger chains (Okazaki pieces, Okazaki et al., 1968a) arise through joining of extremely short polynucleotide chains in a process resembling crystallization rather than unidirectional chain elongation.     

Absorption,Translocation and Metabolism of Nicotinamide in Some Plants

The seedlings of rice, eggplant and tomato at the 5th leaf stage of growth readily absorbed exogenous ¹⁴C-nicotinamide through the root and the foliage in water culture. Within the 24 hr period after the bigining of cultivation, the radioactivity gradually translocated from the part treated with ¹⁴C-nicotinamide to the whole plant body. This compound was rapidly metabolised in the plants to at least six metabolites, in which three compounds were identified as nicotinic acid, NAD and NADP. ¹⁴C-Nicotinic acid was also taken up quickly through the root of rice and its metabolism showed a similar pattern to that of ¹⁴C-nicotinamide. The incorporation of radioactivity into NAD and NADP from ¹⁴C-nicotinamide added to cultivating solution at a concentration of 0.21 ppm was decreased to 10~20% by the simultaneous addition of unlabeled nicotinic acid at a concentration about 1000 times higher than that of the labeled one. It was concluded that the biosynthesis of these pyridine nucleotides from nicotinamide was chiefly via nicotinic acid. The formation of ¹⁴C-nicotinamide in the ¹⁴C-nicotinic acid metabolism suggested a breakdown of NAD. Three unknown compounds observed in both the metabolisms described above were not intermediates in the pyridine nucleotide biosynthesis.     

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance

Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, k_pol and K_d, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (k_pol ∼2.5 s⁻¹) and especially tight nucleotide binding (K_d(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg²⁺ and Mn²⁺. Interestingly, substituting Mn²⁺ for Mg²⁺ accelerated hydrolysis rates >40-fold (k_exo ≥110 s⁻¹ versus ≥2.5 s⁻¹). Preference for Mn²⁺ over Mg²⁺ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families.     

Metabolism of the alpha-Pyridone Ring of Ricinine in Ricinus communis L

Chemically synthesized ricinine-3,5-¹⁴C was used to study the metabolism of this alkaloid in the plant which produces it, Ricinus communis L. In a time course study, ricinine-3,5-¹⁴C was administered to a series of castor plants (Ricinus communis L.) and the radioactivity recovered in the ricinine samples showed a decrease with increase in time. It was also observed that the alkaloid was translocated to the seed. The in vivo conversion of ricinine-3,5-¹⁴C to respiratory ¹⁴CO₂ occurred in both light and dark and indicated that the α-pyridone ring of ricinine could be degraded.     

Storage of nutriments by adult female Glossina morsitans and their transfer to the intra-uterine larva

Administration of U-¹⁴C arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine, or valine into the haemolymph of female Glossina morsitans on the first day of the pregnancy cycle was followed by radiometric analysis of the post-parturient larva. Radioactivity in the larva, expressed as a percentage of the administered activity, was low with histidine (0.3%) and arginine (2.3%) but higher with the other six amino acids (8.2% to 16.8%). ¹⁴C incorporation in the larval lipid was extremely low with arginine and histidine, but with the remaining six amino acids lipids showed the most ¹⁴C labelling. Radioactivity was detected in the larval amino acids corresponding to those injected into the female parents. Further radiometric study using labelled leucine showed that during the first 5 days of pregnancy surplus leucine was largely converted to lipids for larval growth. Thereafter, while the rate of leucine-derived ¹⁴C incorporation in the larval lipids declined rapidly that in the larval proteins increased. Implications are that female G. morsitans has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy destined for larval development, and that normal growth of the intra-uterine progeny is a function of optimum feeding throughout the pregnancy cycle.