The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: III. A Comparison between Some C(3) and C(4) Plants

The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C₃ and C₄ plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light-dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C₄ plants. This light-dependent transfer of label was barely detectable in leaves of C₃ plants and in leaves of F₁ and F₃ hybrids of Atriplex rosea (C₄) and Atriplex patula spp hastata (C₃). The light-dependent transfer of label to photosynthetic products in leaves of C₄ plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-¹⁴C. These experiments, together with the distribution of ¹⁴C in glucose prepared from sucrose-¹⁴C formed during the metabolism of succinate-2,3-¹⁴C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO₂, was the main path of carbon from the cycle to photosynthesis.     

The use of 14C-labeled bacteria as a tracer of ingestion and metabolism of bacterial biomass by microbial grazers

A method for radiolabeling marine bacteria with d-[U-¹⁴C] glucose and a radiotracer method for measuring ingestion and metabolism of bacterial biomass by ciliated protozoa and other microzooplankton are presented. Problems associated with using live bacterial tracers, i.e., label retention, label recycling, tracer cell size and morphology, and intracellular distribution of label are evaluated.Bacterioplankton assemblages collected from field samples incorporated and retained label as efficiently as coastal isolates which were selected for glucose incorporation. Under grazing experimental conditions, labeled bacteria retained a high proportion of the label (hourly net loss = 1.2%). Bacterial recycling of released dissolved organic ¹⁴C (DO¹⁴C) was 0–2.2% of total ¹⁴C per h. Addition of labeled assemblages to nearshore water samples did not detectably alter mean cell size or size frequency distribution of the attendant bacterioplankton assemblages.In grazing experiments with cultured ciliates (Euplotes sp. and Uronema sp.), radioassay parameters varied as direct functions of predator and prey concentrations. Microautoradiographic analysis corroborated that ¹⁴C incorporation measured in the radioassay by filtration techniques primarily represented ingested bacterial biomass and that problems associated with attached and adsorbed labeled bacteria were minimized. Grazing experiments conducted with bacteria labeled with [U-¹⁴C]glucose yielded ingestion rates comparable to bacteria labeled with [U-¹⁴C]thymidine and additionally provided respiration and exudation rates.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES

The effect of O₂ and pH on the in vitro synthesis of ¹⁴C-labeled ureides from [8-¹⁴C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O₂ concentrations and low pH, ureide synthesis was inhibited and the ¹⁴C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O₂ concentrations and high pH, also stimulated ureide synthesis, and the ¹⁴C label was incorporated principally into allantoin. The overall response of the system to O₂ concentration and pH indicated that the per cent distribution of total ¹⁴C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-¹⁴C]hypoxanthine was also inhibited.     

Plant Glycoprotein biosynthesis Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-d-[¹⁴C₁]glucose from uridine diphosphate 2-acetamido-2-deoxy-d-[¹⁴C₁]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation.Hydrolysis of the reaction product yielded glucosamine as the only radiolabeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-d-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100°C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease.     

The fate of [14C]glucose during cold-hardening in Eurosta solidaginis (Fitch)

Glucose catabolism in overwintering larvae Eurosta solidaginis was examined to determine the relative contributions of glycolysis and the pentose phosphate pathway to polyol synthesis at different temperatures. Rates of ¹⁴CO₂ evolution were determined after injection of [¹⁴C]1-glucose, [¹⁴C]6-glucose, and [¹⁴C]3,4-glucose. In addition incorporation of label from each isotope into sorbitol and glycerol was monitored. The respirometric studies showed a relative increase in pentose phosphate activity between 10 and 5°C. Similar results were obtained from the changes of radioactivities incorporated into glycerol, although the activation of the pentose phosphate pathway was low. The conversion of [¹⁴C]glucose to glycerol was highest at 10°C, suggesting that maximum glycerol synthesis may occur at this temperature. Radioactivity appeared in the sorbitol fraction of larvae incubated at temperatures below 5°C. Late autumn larvae converted more [¹⁴C]glucose than did early autumn larvae.     

Evidence for diffusion being the mechanism of translocation in the hyphae of three molds

Translocation of nutrients from a part of the mycelium exposed to an ample supply of a particular nutrient to other parts of the mycelium might be of the utmost importance for the proliferation of fungi in a nutritionally heterogeneous environment. The extent and mechanism of such translocation for saprophytic molds are poorly understood. In this study, diffusion has been shown to be responsible for the translocation of label added as [¹⁴C]glucose and [³²P[orthophosphate to cultures ofRhizopus nigricans grown on opposing gradients of glucose and other nutrients in glass fiber filters. Translocation of the labeled nutrient was found when the label was added to the side of the mycelium that was exposed to high levels of the nutrient. When added to the inoculation point or to the side of the colony exposed to low levels of the nutrient, the labeled compound was immobilized by the mycelium at the addition point. Translocation was bidirectional in that¹⁴C was translocated simultaneously in the opposite direction to³²P. A sink for the translocated nutrients was apparent in the region of sporulation. This pattern of translocation of label added as [¹⁴C]glucose to the glucose surplus side was similar forTrichoderma viride andStemphylium sp.     

Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Patterns of carbon flow from glucose to methane in a thermophilic anaerobic bioreactor

[U-¹⁴C]Glucose, added carrier-free to sludge from a thermophilic anaerobic bioreactor being fed a lignocellulose waste, was rapidly turned over with less than one-third of the original radiolabel remaining as glucose after 5 s of incubation. The primary labeled products found were [¹⁴C]acetate and ¹⁴CO₂, which were in a ratio near 2:1. Further incubation resulted in the disappearance of [¹⁴C]acetate and the appearance of an equivalent amount of label as ¹⁴CH₄ and ¹⁴CO₂. No significant production of [¹⁴C]propionate, butyrate, lactate, or ethanol was detected from [¹⁴C]glucose, even if these potential intermediates (unlabeled) were added to the sludge at a concentration of 1 mM to trap any label entering their pools. Addition of 0.8 atm (80 kPa) of H₂ to the headspace over sludge resulted in some accumulation of [¹⁴C]lactate and a corresponding decrease in [¹⁴C]acetate produced from [¹⁴C]glucose. Production of [¹⁴C]propionate, butyrate and ethanol were still not significant in the presence of H₂. Incubation of sludge for 1 h in the presence of hydrogen resulted in increases in the lactate and formate concentrations, but not those of propionate, butyrate, or ethanol. These results demonstrate that glucose was metabolized directly to acetate, CO₂, and H₂ by the microbial populations in the bioreactor with little carbon from glucose flowing through other intermediates, indicating a high degree of coupling between glucose fermentation and hydrogen uptake. The short-term response of these microbial populations to elevated H₂ partial pressures was to increase lactate production.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Tracer Analysis of Methanogenesis in Salt Marsh Soils

Differences in paths of carbon flow have been found in soils of the tall (TS) and short (SS) Spartina alterniflora marshes of Sapelo Island, Ga. Gaseous end products of [U-¹⁴C]glucose metabolism were ¹⁴CO₂ and ¹⁴CH₄ in the SS region and primarily ¹⁴CO₂ in the TS region. Sulfate concentration did not demonstrably affect glucose catabolism or the distribution of end products in either zone. [U-¹⁴C]acetate was converted to ¹⁴CO₂ and ¹⁴CH₄ in the SS soils and almost exclusively to ¹⁴CO₂ in the TS soils. Sulfate concentration did not affect acetate metabolism in the SS soils; however, a noticeable effect of sulfate dilution was seen in TS soils. Sulfate dilution in TS samples resulted in increased methane formation. Total glucose and acetate metabolism were similar in TS and SS soils despite differences in end products. A microbial community characterized by fermentative/sulfate-reducing processes has developed in TS soils as opposed to the fermentative/methanogenic/sulfate-reducing community found in SS soils.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

Validation of the design of feeding experiments involving [(14)C]substrates used to monitor metabolic flux in higher plants

The aim of this study was to examine whether flux through the pathways of carbohydrate oxidation is accurately reflected in the pattern of ¹⁴CO₂ release from positionally labelled [¹⁴C]substrates in conventional radiolabel feeding studies. Heterotrophic cell suspension cultures of Arabidopsis thaliana were used for this work. The presence of an alkaline trap to capture metabolically generated ¹⁴CO₂ had no significant effect on the ratio of ¹⁴CO₂ release from specifically labelled [¹⁴C]substrates, or on the metabolism of [U-¹⁴C]glucose by the cells. Although the amount of ¹⁴CO₂ captured in a conventional time-course study was only about half of that released from a sample acidified at an equivalent time point, the ratios of ¹⁴CO₂ released from different positionally labelled [¹⁴C]glucose and [1-¹⁴C]gluconate were the same in untreated and acidified samples. Less than 5% of radioactivity supplied to the growth medium as [¹⁴C]bicarbonate was incorporated into acid-stable compounds, and there was no evidence for appreciable reassimilation of ¹⁴CO₂ generated intracellularly during oxidation of [1-¹⁴C]gluconate by the cells. It is concluded that the ratio of label captured from specifically labelled [¹⁴C]glucose is a valid and convenient measure of the relative rates of oxidation of the different positional carbon atoms within the supplied respiratory substrate. However, it is argued that failure to compensate for the incomplete absorption of ¹⁴CO₂ by an alkaline trap may distort estimates of respiration that rely on an absolute measure of the amount of ¹⁴CO₂ generated by metabolism.     

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

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.     

Non-uniform labelling of geraniol biosynthesized from 14CO2 in Pelargonium graveolens

Geranium (Pelargonium graveolens) cuttings were exposed to a 2 hr pulse of ¹⁴CO₂ then allowed to metabolize the label in circulating air for an additional 22 hr. Geraniol isolated from cuttings 2, 4, 8, 12, 16, 20 and 24 hr after the start of the experiment revealed the label in this compound to suffer substantial turnover. Chemical degradation of the labelled geraniol to yield the C₃-isopropylidene fragment showed the distribution of label favored the isopentenyl pyrophosphate-derived half of the molecule. Between 2 and 12 hr of the time-course the distribution of label between the halves of the molecule showed the proportion of label associated with the isopentenyl pyrophosphate-derived half to increase to 78 %. From 12 to 24 hr this preferential labelling declined and approached an equal distribution between the halves. Hypotheses presented to rationalize these observations include the existence of a dimethylallyl pyrophosphate pool and multiple compartments of isoprenoid biosynthesis.     

Determination of Glucose Utilization Rates in Cultured Astrocytes and Neurons with [<Superscript>14</Superscript>C]deoxyglucose: Progress,Pitfalls, and Discovery of Intracellular Glucose Compartmentation

2-Deoxy-d-[¹⁴C]glucose ([¹⁴C]DG) is commonly used to determine local glucose utilization rates (CMR_glc) in living brain and to estimate CMR_glc in cultured brain cells as rates of [¹⁴C]DG phosphorylation. Phosphorylation rates of [¹⁴C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [¹⁴C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMR_glc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [¹⁴C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [¹⁴C]DG distribution space fell at the lowest glucose levels. Calculated CMR_glc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [¹⁴C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.     

Dark Fixation of CO(2) by Crassulacean Plants: Evidence for a Single Carboxylation Step

Malic acid isolated from Bryophyllum pinnatum (Lamk.) Oken (B. calycinum Salisb.), Bryophyllum tubiflorum Harv., Kalanchoë diagremontiana Hamet et Perrier and Sedum guatamalense Hemsl. after dark ¹⁴CO₂ fixation was degraded by an in vitro NADP-malic enzyme technique. In the short term (5 to 30 seconds) the malic acid was almost exclusively labeled in the C-4 carboxyl carbon (greater than 90%). The percentage of ¹⁴C in the C-4 carboxyl of malic acid declined slowly with time, reaching 70% in B. tubiflorum and 54% in B. pinnatum after 14 hours of exposure to ¹⁴CO₂. It was found that malic acid-adapted Lactobacillus arabinosus may seriously underestimate the C-4 carboxyl component of label in malic acid-¹⁴C. The amount of substrate which the bacteria can completely metabolize was easily exceeded; there was a significant level of randomization of label even when β-decarboxylation proceeded to completion, and in extended incubation periods, more than 25% of label was removed from malic acid-U-¹⁴C. The significance of these findings in relation to pathways of carbohydrate metabolism and malic acid synthesis in Crassulacean acid metabolism is discussed.     

Root exudate components change litter decomposition in a simulated rhizosphere depending on temperature

The release of root exudates into the rhizosphere is known to enhance soil biological activity and alter microbial community structure. To assess whether root exudates also stimulated litter decomposition, in a rhizosphere model system we continuously injected solutions of glucose, malate or glutamate through porous Rhizon^® soil solution samplers into the soil at rhizosphere concentrations. The effect of these substances on the decomposition of ¹⁴C-labelled Lolium perenne shoot residues present in the soil was evaluated by monitoring ¹⁴CO₂ evolution at either 15°C or 25°C. The incorporation of the ¹⁴C into the microbial biomass and appearance in the dissolved organic matter (DOM) pool was estimated after 32 d incubation. The presence of malate and glutamate increased the mineralization of L. perenne residues by approximately 20% relative to the soil without their addition at 15°C, however, no significant effects on residue decomposition were observed at 25°C. The incorporation of the ¹⁴C-label into the microbial biomass and DOM pool was not affected by the addition of either glucose, malate or glutamate. Although nearly the same amount of L. perenne residues were mineralized at either temperature after 32 d, less ¹⁴C was recovered in the microbial biomass and DOM pools at 25°C compared to 15°C. Alongside other results, this suggests that the rate of microbial turnover is greater at 25°C compared to 15°C. We conclude that the addition of labile root exudate components to the rhizosphere induced a small but significant increase on litter decomposition but that the magnitude of this effect was regulated by temperature.