Metabolism of [14C]ss-glucose and [14C]-acetate by lettuce embryos during early germination

The metabolism of ['⁴C]-labelled glucose and acetate has been investigated during the early germination - before radicle emergence - of lettuce ( Lactuca sativa L., cv. Val d'Orge) embryos. Similar amounts of radioactivity from both substrates were evolved as C., or incorporated into organic acids, amino acids and proteins. A large part of the [¹⁴C]-glucose was also incorporated into sucrose and polysaecharides, and a small part into the glycerol moiety of lipids. Acetate was massively incorporated into lipids, and only slightly into neutral compounds. These results show that both glucose and acetate can be utilized as respiratory substrates during early germination of lettuce embryos. Various biosynthetic pathways leading to amino acids, proteins, polysaecharides and lipids are active during this period.     

Production of [14C]Acetylcholine and [14C]Carbon Dioxide from [U–14C]Glucose in Tissue Prisms from Aging Rat Brain

Abstract: Production of [¹⁴C]acetylcholine and ¹⁴CO₂ was examined by using tissue prisms from neocortex, hippocampus, and striatum from rats aged approximately 5 months, 13 months, and 27 months. [¹⁴C]Acetylcholine synthesis in the striatum showed highly significant decreases with age for measurements in the presence of both 5 m m - and 31 m m -K⁺, contrasting with the lack of significant change in ¹⁴CO₂ production in this region. The neocortex and hippocampus showed only small changes, especially when comparison was made between 13-month and senescent animals. Measurements of the release of [¹⁴C]acetylcholine and influence of atropine on this release confirmed the relative stability with age of the cholinergic system in the neocortex.     

[14C]GLUTAMATE UPTAKE AND COMPARTMENTATION IN GLIA OF RAT DORSAL SENSORY GANGLION

Abstract— The characteristics of the uptake of l -[U-¹⁴C] glutamate into rat dorsal sensory ganglia were investigated. The uptake was mediated by two distinct kinetic systems, with apparent K_m values of the order of 10⁻³ M (low affinity) and 10⁻⁵ m (high affinity). The high affinity uptake system was strongly dependent upon temperature and sodium ion concn, and was depressed by a number of metabolic inhibitors. Following uptake, [¹⁴C] glutamate was extensively metabolized, primarily to glutamine, although this was not so with cultured ganglia, where in addition to an increased uptake of [¹⁴C] glutamate, the specific radioactivity of glutamate was increased and that of glutamine decreased. The labelled substrates [U-¹⁴C]pyruvate and [U-¹⁴C] acetate were used to investigate this phenomenon and the results are discussed in relation to current knowledge of metabolic compartmentation in nervous tissue.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

METABOLISM OF [14C]LEUCINE AND [14C]ACETATE IN SENSORIMOTOR CORTEX, THALAMUS, CAUDATE NUCLEUS AND CEREBELLUM OF THE CAT

Abstract— —In the head of the caudate nucleus, the relative specific activity of glutamine (glutamic acid specific activity = 1) was less than 1 with intravenous [¹⁴C]leucine as the tracer metabolite. This is in contrast to observations made in other brain areas (cortex, hippocampus, thalamus, pons, and medulla) where the relative specific activity of glutamine was greater than 1. This is also in contrast to findings when [l-¹⁴C]acetate was utilized as the tracer; under these conditions, in all brain areas, including the head of the caudate nucleus, the relative specific activity of glutamine was greater than 1. It is inferred that the differences in metabolism of [¹⁴C]leucine and [¹⁴C]acetate in the head of the caudate from that in other brain areas reflect differences in compartmentation of the glutamate-glutamine system.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

Photosynthetic 14CO2 fixation and [15N]-ammonia assimilation during UV-B radiation of Lithodesmium variabile

Uptake of [¹⁵N]-ammonia was more sensitive to UV-B exposure than the total ¹⁴CO₂ fixation rate of Lithodesmium variabile Takano. Short-term UV-B radiation (15 min) had practically no effect on the kinetics of [¹⁵N]-ammonia, whereas there was an effect on [¹⁴C]-bicarbonate uptake rate. A significant reduction was found after 30 and 60 min UV-B stress. The time course of photosynthetic uptake of ¹⁵NH₄Cl at several wavelengths was markedly depressed at shorter wavelengths (irradiation with WG 280). A short-term (11 min) exposure to ultraviolet radiation had no influence on the [¹⁴C]-labeled photosynthetic products. However, the [¹⁵N]-label of several amino acids and the ratio of [¹⁵N]-glutamine to [¹⁵N]-glutamic acid varied after irradiation with different ultraviolet wavebands. The results are discussed with reference to UV damage to the key enzymes of nitrogen metabolism.     

METABOLISM OF d-[U-14C]RIBOSE IN RAT TISSUES

Abstract— d -[U-¹⁴C]Ribose injected subcutaneously into the rat enters the blood, liver and brain. At 30 min after injection 40-70 per cent of the radioactivity in the brain was found in amino acids and only 2-6 per cent in free sugars. In contrast, free sugars (mainly glucose) and carboxylic acids accounted for most of the radioactivity in liver and blood. Evidence for the entry of [U-¹⁴C]ribose into the brain was obtained by intracarotid or intravenous injection of [U-¹⁴C]ribose after interrupting the blood supply to the liver and kidney. Under these conditions the radioactivity in the brain was found in amino acids, carboxylic acids and ribose; no significant amount of [¹⁴C]glucose was detected in brain or heart. It is concluded that ribose is metabolized directly in vivo in the brain. d -[U-¹⁴C]Ribose was metabolized also by brain slices in vitro to form ¹⁴C-labelled amino acids and carboxylic acids; the rate was equivalent to the utilization of 0.65 μ mol of ribose/g/h. The specific radioactivity of glutamine and of γ -aminobutyrate was similar to or higher than that of glutamate in the brain. These results are discussed in the context of metabolic compartments.     

Synthesis and proof-of-function of a [14C]-labelled form of the plant iron chelator nicotianamine using recombinant nicotianamine synthase from barley

The amino acid nicotianamine (NA) is essential for micronutrient metabolism in plants. Lack of NA results in a chlorotic phenotype and oxidative stress, since NA is a chelator of iron and other metal nutrients. To investigate the precise cellular function of NA in micronutrient transport and homeostasis, a protocol for the production of [¹⁴C]-labelled NA was developed. Recombinant NA synthase was used to generate [¹⁴C]-NA from [¹⁴C]- S -adenosylmethionine. After purification by solid-phase ion exchange about 66% yield was achieved. The identity of the [¹⁴C]-NA with chemically synthesized NA was demonstrated by several independent methods, including two TLC systems, two HPLC systems and immuno-detection. Moreover, biological function was shown by complementation of the Lycopersicon esculentum mutant chloronerva that is free of NA due to a defect in NA synthase. Proof-of-function for the produced [¹⁴C]-NA as a suitable tool for transport studies was provided monitoring the distribution of [¹⁴C]-NA after feeding to tomato and Ricinus communis seedlings.     

Metabolism of [14C]-indole-3-acetic acid by soybean callus and hypocotyl sections

Metabolism of indole-3-acetic acid in soybean [ Glycine max (L.) Merr.] was investigated with [1-¹⁴C]- and [2-¹⁴C]-indole-3-acetic acid (IAA) applied by injection into soybean hypocotyl sections and by incubation with soybean callus. Free IAA and its metabolites were extracted with 80% methanol and separated by high performance liquid chromatography with [³H]-IAA as an internal standard. Metabolism of IAA in soybean callus was much greater than that in tobacco ( Nicotiana tabacum L.) callus used for comparison. High performance liquid chromatography of soybean extracts showed at least 10 metabolite peaks including both decarboxylated and undecarboxylated products. A major unstable decarboxylated metabolite was purified. [¹⁴C]-indole-3-methanol (IM) was three times more efficient than [2-¹⁴C]-IAA as substrate for producing this metabolite. It was hydrolyzable by β-glucosidase (EC 3.2.1.21), yielding an indole and D-glucose. The indole possessed characteristics of authentic IM. Thus, the metabolite is tentatively identified as indole-3-methanol-β-D-glucopyranoside. The results suggest that soybean tissues are capable of oxidizing IAA via the decarboxylative pathway with indole-3-methanol-glucoside as a major product. The high rate of metabolism of IAA may be related to the observed growth of soybean callus with high concentrations of IAA in the culture medium.     

Studies on the uptake of (14C) amino acids derived from both dietary (14C) protein and dietary (14C) amino acids by rainbow trout, Salmo gairdneri Richardson

Previous studies have shown that rainbow trout fed on diets containing whole protein have superior growth rates compared to fish fed on diets of similar amino acid composition but containing a high proportion of free amino acids. The influence of several nutritional factors on the uptake of radioactivity from food pellets containing either [U-^I4C] protein or [U-¹⁴C] amino acids into the systemic blood of trout has been investigated. The time taken for radioactivity in the free amino acid fraction of blood to reach a peak after a meal containing [U-¹⁴C] protein had been given was much shorter, and the level of radioactivity in the blood higher, in trout with almost empty stomachs than in fish with almost full stomachs; uptake of radioactivity into blood amino acids was also more rapid and reached much higher concentrations when pellets containing [U-¹⁴C] amino acids were fed than when [U-¹⁴C] protein was fed. Incorporation of radioactivity into blood protein continued for a much longer period and reached higher levels when a pellet containing [U-¹⁴C] protein was fed than when a pellet containing [U-¹⁴C] amino acids was fed. Previous dietary history (low or high protein intake) did not appear to affect the rate of absorption of amino acids from either protein or free amino acid pellets. The uptake rates from pellets containing free amino acids could be slowed by mixing the dietary amino acids with albumin. The distribution, postabsorption, of radioactivity in the different fractions of blood and liver suggested that incorporation of carbon residues into glycogen and lipid from an amino acid diet was greater than from a protein diet. The converse was true of incorporation of radioactivity into tissue protein.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

Incorporation of 15N and 14C of methionine into the mugineic acid family of phytosiderophores in iron-deficient barley roots

The role of methionine as a precursor in mugineic acid (MA) biosynthesis was studied by feeding ¹⁵N-ammonium sulfate, ¹⁴C-amino acids, and [1-¹⁴C, ¹⁵N]-methionine to iron-deficient barley roots ( Hordeum vulgare L. cv. Minorimugi), grown hydroponically. The incorporation of isotopes into amino acids was also examined. Methionine appears to be the most efficient precursor of the mugineic acid family (MAs) of phytosiderophores; homoserine was also incorporated into the MAs, but other amino acids such as glutamate, alanine, and γ-amino butyric acid did not act as precursors of MAs. Carbon-14 and ¹⁵N of methionine were incorporated into MAs. This specific incorporation of ¹⁴C and ¹⁵N indicated that the nitrogen atoms of MAs were derived from two molecules of methionine. It is suggested that deoxymugineic acid (DMA) is probably the first phytosiderophore to be synthesized on the biosynthetic pathway of MAs.     

Analysis of [14C] indole-3-acetic acid metabolites from the primary roots of Zea mays seedlings using reverse-phase high-performance liquid chromatography

Methanolic extracts of Zea mays L. cv. Fronica root segments which had been incubated in [¹⁴C] indole-3-acetie acid were analysed by reverse-phase high-performance liquid chromatography. Metabolism of indole-3-acetic acid was found to be rapid and extensive with at least 11 products apparent after a 2 h incubation. A comparison of metabolites of [1-¹⁴C]– and [2-¹⁴C] IAA, calculations of ¹⁴CO₂ evolution, and data on the polarity of products indicated that decarboxylation had not occurred. An average of 34% of the radioactivity remained associated with the indole-3-acetic acid peak.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

UPTAKE OF [14C]NIPECOTIC ACID INTO RAT DORSAL ROOT GANGLIA

Abstract— [¹⁴C]Nipecotic acid was accumulated in isolated desheathed rat dorsal root ganglia by a saturable process with K _m= 48.8 μ m and V _max= 2.2 nmol/g/min. The concentration of l -2.4-diamino-butyric acid required to inhibit the uptake of nipecotic acid by 50% was three times the concentration of β-alanine required to do the same. Light microscopic autoradiography indicated that the sites of uptake of [¹⁴C]nipecotic acid were principally confined to satellite glial cells. It is concluded that nipecotic acid is transported by the GABA uptake system in glia but that it has less affinity for this system than GABA.     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.