The fate of carbon during the assimilation of carbamoyl phosphate in white spruce seedlings as revealed by [14C]-carbamoyl phosphate, [14C]-cyanate, and [14C] -bicarbonate labeling patterns

The fate of carbamoyl phosphate in white spruce seedlings revolves around the production of spontaneous degradation products, cyanate, bicarbonate, and carba-mate. When [¹⁴C]-carbamoyl phosphate and [¹⁴C]-cyanate are assimilated, urea is a common early metabolic intermediate that appears in the alcohol soluble N. By contrast, urea is not detected among the products of [¹⁴C]-bicarbonate. Carbamoyl phosphate and glutamic acid are implicated as having pivotal roles in the production of amides, arginine, and biotin. Within 2-h exposures to radioactive substrates considerably more carbon from bicarbonate was diverted into amino acids Incorporated into proteins than with carbon-nitrogen substrates. Specific activities of bound amino acid residues support the view that proteins formed from these [¹⁴C]-substrates have different rates of metabolic turnover.     

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.     

[14C]AMINO ACID FORMATION FROM LABELLED GLUCOSE AND/OR ACETATE IN BRAIN CORTEX SLICES WITH EXPERIMENTALLY ELICITED PROLIFERATION OF ASTROGLIA. CORRELATION OF BIOCHEMICAL AND MORPHOLOGICAL CHANGES

Abstract— Changes in morphology and in transformations of [U-¹⁴C]glucose and [1-¹⁴C]acetate into amino acids of the brain cortex were followed on the Sth, 10th and 21st days after production of mechanical lesions and compared with control tissue. In the experimental tissue, proliferation of astroglia and reduction of the number of neurons had taken place. On the 10th day, accumulation of mitochondria and of some gliofilaments in the cytoplasm of astroglia was observed. On the 21st day, the gliofilaments occupied a substantial portion of the astroglial cytoplasm and the mitochondria were reduced in number and compressed to the cell membrane. Incorporation of ¹⁴C from acetate into amino acids was substantially increased on the 10th day (up to 240% with respect to controls) and normalized again on the 21st day. Incorporation of [¹⁴C]glucose into amino acids decreased somewhat during the experimental period. It has been proposed that the proliferation of astrocytes and their ultrastructural changes may account for the increased transformation of [¹⁴C]acetate into amino acids, in particular into glutamine which is formed from the small glutamate pool.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

Effect of Stimulation on the Incorporation of 14C from Glial and Neuronal Specific Substrates into Brain Proteins In Vivo and In Vitro

Abstract: The incorporation of amino acids into brain proteins following brachial plexus stimulation (BPS) was studied in anaesthetised Sprague-Dawley rats following injection of radioactive precursors of both neuronal and glial compartments. Following intraperitoneal injection of [¹⁴C]glucose, which is the major neuronal pool precursor, BPS resulted in a significant increase of 379% ( P ± 0.001) in the incorporation of carbon from [¹⁴C]glucose into TCA-insoluble proteins in the contralateral sensorimotor cortex as compared with the ipsilateral area of the same animal. This increase was abolished totally when tetrodotoxin (10 μg ml^-1) was applied topically to the surface of the stimulated area. Following intraperitoneal injection of [¹⁴C]acetate, which is considered to be mainly a glial cell precursor, the same afferent electrical stimuli caused a significant decrease of 21% in the incorporation of amino acids into proteins in the stimulated versus unstimulated sensorimotor cortex. With [4-³H]phenyl-alanine or [l-¹⁴C]leucine as precursors a significant decrease (12%) or no change was recorded, respectively. A similar decrease in protein synthesis in the stimulated sensorimotor cortex was achieved using different routes of injection. No significant changes were observed in the ratio of the specific radioactivities of the total amino acids of the two hemispheres using either precursor. In vitro , synaptosomes showed a large increase in incorporation into proteins after treatment with electrical pulses, both with [¹⁴C]glucose and with [U-¹⁴C]acetate as precursors.     

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.     

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.     

[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.     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

Quantitative aspects of triacylglycerol metabolism and sterol synthesis from 14C-acetate in etiolated seedlings of Euphorbia lambii

Triacylglycerols occur in both the endosperm and embryo of Euphorbia lambii seeds. Upon germination, the amount of these neutral lipids in the endosperm decreased with 1.06 mg fatty acid day^-1. The embryo contained 1.4 mg fatty acids in the triacylglycerols and this value declined slowly to 0.4 mg seedling^-1 during the 8 day period of endosperm depletion. Radioactive acetate was rapidly taken up by the cotyledons of intact seedlings, translocated throughout the entire seedling, and up to 10.5% of the ¹⁴C proceeded to the sterols and latex triterpenols. Maximum uptake values of 1.4 μmol seedling^-1 day^-1 of acetate were measured. Acetate uptake and subsequent incorporation into sterols and triterpenols decreased substantially in the presence of increasing amounts of sucrose (up to 0.3 M). Traces of acetate did not effect [¹⁴C]-sucrose uptake and corresponding synthesis of [¹⁴C]-sterols and triterpenols, but increased concentrations of acetate (0.05 M and up) reduced both uptake of sucrose and its conversion into unsaponifiable lipids.
The uptake capacity of the cotyledons for [¹⁴C]-glycerol exceeded the daily production in the endosperm, but only a small amount of label proceeded to the sterols and triterpenols. [¹⁴C]-Triacylglycerols were never detected in the seedling, regardless of the labeled substrate used. Although acetate is an efficient precursor in triterpenol and sterol synthesis, the uptake capacity of the cotyledons for this metabolite is too small in relation to the daily production of water soluble substrates in the endosperm. If acetate is released by the endosperm, only a marginal contribution towards triterpenol and sterol synthesis in the seedling is to be anticipated from this substrate.     

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.     

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.     

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.     

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.     

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.     

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.     

Localized 13C NMR Spectroscopy in the Human Brain of Amino Acid Labeling from d-[1-13C]Glucose

Abstract: Cerebral metabolism of d [1-¹³C]glucose was studied with localized ¹³C NMR spectroscopy during intravenous infusion of enriched [1-¹³C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-¹³C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-¹³C]glutamate clearly lagged behind that of [4-¹³C]glutamate and peaked at t = 110–140 min. Multiplets due to homonuclear ¹³C-¹³C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a ¹³C isotopic fraction at C4 glutamate of 27 ± 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 ± 1%), p < 0.05. By comparison with an external standard the total amount of [4-¹³C]glutamate was directly quantified to be 2.4 ± 0.1 µmol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 ± 0.7 µmol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in healthy human brain.     

Studies on [14C]-glucose metabolism during shoot bud induction in cultured cotyledon explants of Pinus radiata

Bender, L., Joy IV, R. W. and Thorpe, T. A. 1987. Studies on [¹⁴C]-glucose metabolism during shoot bud induction in cultured cotyledon explants of Pinus radiala.
Excised cotyledons of Pinus radiata D. Don, cultured under shoot-forming (plus N⁶-benzyladenine) and elongating (minus N⁶-benzyladenine) conditions, were fed U-[¹⁴C]-glucose for 3 h in the light followed by a 3 h chase period immediately after excision (day 0) and after 3 days of culture (day 3). The incorporation of ^l4C into individual soluble metabolites as well as into protein was followed. No labelled citrate could be detected at day 0, however, a flow of ¹⁴C from glucose to glutamate/ glutamine occurred. During this stage the synthesis of glutamine strongly increased in the cotyledons supplied with N⁶-benzyladenine, which suggests a positive influence of this cytokinin on nitrogen incorporation prior to differentiation. After 3 days of cultivation large amounts of labelled citrate were detected. An increased incorporation of label into protein due to the cytokinin treatment was not detected during the early culture period (days 0 and 3). Labelled amino acids were incorporated into protein to different degrees, but this was not influenced by the hormonal treatment.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.