Inter-comparison of 14C-labelled bicarbonate solutions prepared by different institutes for measurement of primary productivity in natural waters and monoalgal cultures

An intercomparative study was carried out to investigate possibleeffects on primary productivity measurements when using NaH¹⁴C0₃solutions prepared by different methods. Five different ampoulebatches coded A, B, C, D and E were tested. Three of the batches(A, B and D) had been produced by direct dilution of industriallyproduced NaH¹⁴CO₃, of high specific activity. A and D were dilutedwith distilled water added carrier, whereas no information onhow batch B was diluted could be obtained. Batch E was preparedby trapping ¹⁴CO₂, gas — released by strong HCl from Ba¹⁴CO₃— in sodium hydroxide. In the case of batch C, the processof manufacture was not known. The tests were carried out ondifferent phytoplankton material with low algal density. Twobatches (B and C) showed significant inhibitory effects on P_calc(–5–44%), on the slope of the ascending part ofthe light adaptation curve (), and on P_max. Batch A showed minor,but still significant effects. The four batches A, B, C andD carried rather high amounts of non-volatile rest activity(between 13 and 194 d.p.m./µCi), which made measurementsof the release of extracellular dissolved organic carbon (EOC)almost impossible. This phenomenon,per se, would if uncor-rectedproduce considerably higher per cent EOC release in low-productivewaters, as has been reported by many authors. As to the standardizationof the working solutions, two batches (C and D) showed a pooraccuracy (16 and 18% deviation, respectively) when tested atthe C14C, and two batches (A and B) showed unacceptably highvariability between ampoules of the same batch. The study indicatesthat it is not recom-mendable to use working solutions preparedby direct dilution of industrially produced NaH¹⁴CO₃ of highspecific activity without prior testing of possible effectson algal photosynthesis. It is recommended that the specificactivity of the working solution be measured if it is not exactlyspecified by the manufacturer by a ‘Certificate of Quality’.     

Comparison of two methods to measure algel release of dissolved organic carbon and the subsequent uptake by bacteria

Two methods were used to compare phytoplankton release and bacterialuptake of extracellular organic carbon (EOC) products. A sizefractionation and an antibiotic method were used simultaneouslyduring several diel in Situ studies in Danish lakes and onecoastal area. Phytoplankton populations were very sensitiveto even small concentrations of antibiotics (1 µg ml^–1).Generally the bacterial activity was inhibited by 50%. In caseswhere the antibiotics did not affect phytoplankton photosynthesis,the two methods agreed reasonably. Bacterial respiration dataobtained with the antibiotic method were in the range of valuesfrom the literature. This supports the conclusion that the partialuncoupling of EOC release and uptake with antibiotics can beused to follow the flux of EOC in situ. The effect on the phytoplanktonmust however, be evaluated in each experiment. The methodologicalproblems of both approaches are discussed.     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 1. The Time Course of 14CO2 Production

Glucose, either uniformly labelled with¹⁴C, or specificallylabelled in the I, 2, or 6 position, was added to C. vulgaris.Radio-active carbon dioxide was produced initially ten timesfaster from glucose-I-¹⁴C than from glucose-6-¹⁴C. This differencewas found with carbohydrate-starved cultures, exponentiallygrowing cultures, and cultures assimilating ammonia or nitraterapidly. A similar difference was also found with C. pyrenoidosaand Ankistrodesmus. 37 per cent. of the ¹⁴C added as glucose-¹-¹⁴Cto exponentially growing cells was recovered as carbon dioxidebut generally the recovery was less than this. Only 5 per cent.of ¹⁴C added as glucose-6-¹⁴C was recovered as carbon dioxide.The specific activity of the carbon dioxide produced was considerablylower than that of the carbon in the added glucose.     

Physiology of diatom Skeletonema costatum (Grev.) Cleve photosynthetic extracellular release: evidence for a novel coupling between marine bacteria and phytoplankton

The photosynthesis of cellular materials by phytoplankton isaccompanied by release of organic molecules from the algal cellsinto the water. The patterns of carbon fixation in particulateand dissolved pools were investigated in Skeletonema costatumcultured under 12 h light/12 h dark cycles. The short-term production(1–15 min) of particulate organic carbon (POC) and extracellularorganic carbon (EOC) compounds was studied by measuring theuptake of ¹⁴C-labelled sodium bicarbonate and its subsequentincorporation and release into organic compounds. Slightly modifiedtraditional ¹⁴C radiotracer protocols were used, including separationby electrophoresis and thin-layer chromatography and detectionby autoradiography. Results indicated that there was a distinctdifference between radiolabelled compounds in the POC and EOCpools. Several metabolites found in the EOC pool were not presentin the POC pool, indicating the active release of these productsfrom the cells into the ambient water during short-term incubations,and indicating that inorganic carbon fixation pathways in marineautotrophs might be partly extracellular.     

Carbon-specific phytoplankton growth rates: a comparison of methods

Measurements of biomass and growth rate of two axenic algalcultures were carried out using three different methodologicalapproaches: the specific ¹⁴C-labelling of chlorophyll a, [³H]adenineincorporation into DNA and net organic carbon assimilation.Time-course experiments revealed that the specific activitiesof chlorophyll a were significantly higher than the specificactivity of total algal carbon in six of seven experiments.When the specific activity of chlorophyll a is used to calculatethe carbon biomass and growth rate, the carbon biomass of thealgae will thus be underestimated and the specific growth ratewill be too high. Determination of growth rates from incorporationof [³H]adenine gave lower values than those obtained from netorganic carbon assimilation and from ¹⁴C incorporation intochlorophyll a. Problems with adenine saturation are suggested.When [³H]adenine is used to measure growth rates in dense algalcultures, additions of >1 µM [³H]adenine are oftenrequired to maximally label the extracellular and intracellularadenine pools and hence DNA.     

Carbon Loss from the Roots of Forage Rape (Brassica napus L.) Seedlings Following Pulse-labelling with 14CO2

The loss of organic material from the roots of forage rape (Brassicanapus L.,) was studied by pulse-labelling 25-d-old non-sterilesand-grown plants with ¹⁴CO₂. The distribution of ¹⁴C withinthe plant was measured at 0, 6 and 13 d after labelling whilst¹⁴ C accumulating in the root-zone was measured at more frequentintervals. The rates of ¹⁴C release into the rhizosphere, andloss of ¹⁴CO₂ from the rhizosphere were also determined. Thesedata were used to estimate the accumulative loss of ¹⁴C fromroots and loss respiratory ¹⁴CO₂ from both roots and associatedmicro-organisms. Approximately 17-19% of fixed ¹⁴CO₂ was translocatedto the roots over 2 weeks, of which 30-34% was released intothe rhizosphere, and 23-24% was respired by the roots as ¹⁴CO₂. Of the ¹⁴C released into the rhizosphere, between 35-51%was assimilated and respired by rhizosphere micro-organisms.Copyright1993, 1999 Academic Press Brassica napus L., carbon loss, carbon partitioning, microbial nutrition, microbial respiration, forage rape, pulse-labelling, rhizodeposition, root respiration, sand culture     

Regulation of bacterial secondary production in two eutrophic lakes and in experimental enclosures

Bacterial cell number and secondary production of bacterioplanktonwere measured during two periods of 14 and 19 days, respectively,in experimental enclosures of lake water (each containing about5–7 m³ and in two Danish, eutrophic lakes. Additions ofphosphate and planktivorous fish to some of the enclosures inducedrapid changes in the phytoplankton biomass, primary productionand release of extracellular organic carbon (EOC). In some ofthe enclosures containing fish, release of EOC from phytoplanktoncon stituted from 36 to 55% of the bacterial secondary productiondetermined by means of [³H]thymidine incorporation into DNA.In others, such a relationship was not found, suggesting thatphytoplankton species-specific release rates of compositionof EOC might play a role in the microbial response. In enclosureswithout fish, released dissolved organic carbon from zooplanktongrazing activities could Sustain     

Storage Pools and Turnover Systems in Growing and Non-growing Cells: Experiments with 14C-Sucrose, 14C-Glutamine, and 14C-Asparagine

¹⁴C-labelled sucrose, glutamine, and asparagine have been suppliedto aseptically cultured carrot explants that either grew rapidlyby cell division or, by contrast, only slowly by cell expansion.The radioactive substrates were supplied in a brief ‘pulse’followed by a much longer period during which the tissues weresupplied with ¹²C-substrates. The passage of ¹⁴C through thevarious soluble compounds of the tissue and into the proteinwas followed. Alternatively, the ¹⁴C-labelled compound was suppliedthroughout the entire period of an experiment while the tissuealso received ¹²C-sucrose. The pulse-labelling experiments demonstrateturnover and the fate of the breakdown products, as well asthe emphasis placed on this kind of metabolism by cells at differentlevels of activity in their growth. The long-term labellingexperiments show the different ways in which carbon from varioussources may be used and how these pathways are affected by growth.The amount of ¹⁴C present in the various free (ethanol soluble)and combined (ethanol insoluble, acid-hydrolysable compounds—proteins)was determined, as well as the specific activity of the carbonin each compound. The fate of ¹⁴C supplied as sucrose had muchin common with ¹⁴C supplied as glutamine, with respect to theease with which it entered both the protein being synthesizedand the carbon dioxide evolved, but it was very different from¹⁴C supplied as asparagine. To interpret these data, compartmentsor pools of metabolites are postulated in the organized cell;exogenous ¹⁴C-sucrose and ¹⁴C-glutamine readily furnish carbonfor pools of amino-acids en route to protein, which are protectedfrom both the stored compounds and those which arise after proteinbreakdown. However, exogenous ¹⁴C-asparagine enters, is accumulated,and persists in the pool of stored compounds which also receivethe nitrogen-rich substances that arise from protein breakdown.The kinetic data and the specific activities of the carbon inits various forms require that protein breakdown and re-synthesisoccur concomitantly, that the stimulus to grow, exerted by coconutmilk, accentuates protein synthesis and also the pace of itsturnover, that some respired carbon dioxide arises from protein,and that this moiety of the respiration is increased by thecoconut-milk stimulus as it accentuates the pace of cyclicalturnover. In similar experiments with free cells from differentplants, the same general conclusions apply, but the rates ofturnover of protein are greater in free cells than in tissueexplants. Some specific differences, however, exist. Cells ofArachis, the only legume investigated, permit ¹⁴C-asparagineto contribute, like ¹⁴C-glutamine, to both protein synthesisand respired ¹⁴CO₂; it is not merely segregated in a storagepool. Thus, by virtue of their organization, plant cells maintainthe same substances simultaneously in distinct phases or compartments,where they play distinctive roles, without mingling. Geneticsendows each cell with the information that makes its biochemicalreactions feasible; the organization of the cells determineshow far the feasible becomes practised in cells in any givensituation.     

Development of Lipid Synthesis from CO2 in Avena Leaves during Greening of Etiolated Seedlings

The development of the lipid synthesizing system in Avena leafsections was examined in connection with carbon fixation duringthe greening of etiolated seedlings under light. During theinitial 2 h illumination there was a low level of CO₂ fixationby PEP carboxylation, but its products, malate and citrate,did not serve as a carbon source for lipid synthesis, althoughlipid synthesis from acetate had already been established. Withthe initiation of Calvin cycle activity after the initial 2h illumination, lipid synthesis began, with CO₂ fixed by RuBPcarboxylation serving exclusively as the carbon source. Fattyacid synthesis in the leaves during the initial 3 h illumination,unlike the fatty acid synthesis thereafter, was insensitiveto thiolactomycin, an inhibitor of type II fatty acid synthetasecontained in the plastids, and was not dependent on light, incontrast to light-dependent activity in greened leaves. The distribution of ¹⁴C incorporated into lipid molecules fromNaH¹⁴CO₃ showed an equal ratio of ¹⁴C in fatty acid, glyceroland choline moieties of labeled phosphatidylcholine, but a denserradioactivity in the galactose moiety than in the residual moietyof mono- and di-galactosyldiacylglycerols. This suggests a regulatedsupply of glycerol, choline and fatty acid moieties for phosphatidylcholinesynthesis, and an excess supply of galactose to diacylglycerolmoiety for galactosyldiacylglycerol synthesis in Avena leaves. (Received October 31, 1984; Accepted January 25, 1985)     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Effect of CO2 Concentration on Growth, Carbon Distribution and Loss of Carbon from the Roots of Maize

The effects of three ranges of CO₂ concentration on growth,carbon distribution and loss of carbon from the roots of maizegrown for 14 d and 28 d with shoots in constant specific activity¹⁴CO₂ are described. Increasing concentrations of CO₂ led toenhancement of plant growth with the relative growth rate (RGR)of the roots affected more than the RGR of the shoots. Between16% and 21% of total net fixed carbon (defined as ¹⁴C retainedin the plant plus ¹⁴C lost from the root) was lost from theroots at all CO₂ concentrations at all times but the amountsof carbon lost per unit weight of plant decreased with time.Possible mechanisms to account for these observations are discussed. Key words: Growth, Roots, Carbon loss, [CO₂]     

Effects of Temperature and Light on the Carbon Budget of Young Cucumber Plants Studied by Steady-State Feeding with 14CO2

Shishido, Y., Challa, H. and Krupa, J. 1987. Effects of temperatureand light on the carbon budget of young cucumber plants studiedby steady-state feeding with ¹⁴CO°₂J. exp. Bot. 38: 1044–1054. The effect of temperature on the fate of ¹⁴C assimilated insteady-state by the expanding third leaf of cucumber seedlingswas studied at irradiances of either 30 or 75 W m^–2 (PAR)with a daylength of 8 h. The irradiance did not affect the relativedistribution of ¹⁴C assimilated by the source leaf between growth,respiration and export. In the range 15–30°C risesin temperature generally increased the proportion of carbonexported. The average rate of carbon exported during the nightwas about half the rate in the day. About 45% of the exportedcarbon was lost by respiration. The distribution pattern ofcarbon exported during the day differed considerably from thatof carbon exported during the night. The intensity of irradiance did not affect the proportion oflabelled carbon recovered from the roots. Thus the decreasedshoot/root ratio generally observed with increased irradianceis not directly controlled by carbohydrate supply. We found that the distribution patterns of exported ¹⁴C do notnecessarily represent the real carbon distribution, due to differencesin specific activity of imported carbon of individual organs.Consequently distribution patterns of ¹⁴C observed in experimentswith one source leaf have to be considered with caution. Key words: Carbon budget, ¹⁴C, ¹⁴C steady-state feeding, translocation, respiration, assimilate distribution, cucumber, temperature     

The Translocation of 14C-Photoassimilate from Normal and Mutant Leaves to the Pods of Pisum sativum L.

In experiments using radioactive carbon dioxide (¹⁴CO₂) a comparisonwas made of the ¹⁴C-photoassimilate translocation potentialsof two normal leaved (genotype AfAfTlTl) and two mutant formsof Pisum sativum (pea). A ¹⁴CO₂ administration method is describedthat permitted ¹⁴C-translocation studies to be conducted underfield conditions. One of the mutants available produced tendrils in place of leaves(afafTlTl). The other mutant studied was without tendrils buthad a much branched petiole with numerous relatively minuteleaflets (afaftltl). These mutants and the normal-leaved cultivarswith which they were compared were not isogenic lines. Lengthybackcrossing would be required before full assessment couldbe made of the possible agronomic value of such mutations. An interim evaluation of these mutants was based on ¹⁴C-distributionassays that were conducted 48 h after feeding ¹⁴CO₂, to specifiedleaves. The indication was that in translocation terms the leafand pod had a well defined respective source and sink relationshipthat was independent of leaf morphology. In each case the podswhich constituted the major ¹⁴C sinks depended on which leafhad been fed ¹⁴CO₂. With regard to sink specific activity asdefined by the quantity of ¹⁴C incorporated per unit dry weightof pod, the mutants were not significantly different from normal. The implication of these findings was that fundamental changesin pea leaf morphology could be made genetically without a markedeffect on the photoassimilate export potential of the leaf.     

Variation in 14C Partitioning in the Tips of Growing Stolons of Potato (Solanum tuberosum L.)

¹⁴C partitioning was examined in growing stolons of field-grownpotato (Solanum tuberosum L.) cv. Maris Piper. Considerablevariation was evident on single plants and on a fresh weightbasis many stolon tips, which showed no signs of sub-apicalswelling, had higher specific activities (cpm g^–1 f. wt)of ¹⁴C in both ethanol soluble and insoluble forms than larger,visibly tuberized stolons. Furthermore, many tips of low freshweight had a higher insoluble to soluble ¹⁴C ratio than visiblytuberized stolons suggesting greater efficiency of conversionof soluble ¹⁴C to insoluble ¹⁴C in the smaller stolons. Theresults suggest that the onset of visible ‘tuberization’,namely the sub-apical swelling of the stolon, is preceded byincreased soluble carbon accumulation at the stolon tip togetherwith an increase in the conversion of soluble to insoluble formsof carbon. Tuberization, ¹⁴C, stolon tip     

The Distribution and Identity of Assimilates in Tomato with Special Reference to Stem Reserves

Much of the work on the distribution of ¹⁴C-labelled assimilatesin tomato has been done in winter under low light intensities,and consequently the reported distribution patterns of ¹⁴C maynot be representative of plants growing in high light. Further,there are several somewhat conflicting reports on patterns ofdistribution of ¹⁴C-assimilates in young tomato plants. We soughtto clarify the situation by studying the distribution of ¹⁴C-assimilatesin tomato plants of various ages grown in summer when the lightintensity was high. In addition, the role of the stem as a storageorgan for carbon was assessed by (a) identifying the chemicalfractions in the stem internode below a fed leaf and monitoring¹⁴ C activity in these fractions over a period of 49 d, and(b) measuring concentrations of unlabelled carbohydrates inthe stem over the life of the plant. The patterns of distribution of ¹⁴C-assimilates we found fortomato grown under high light intensity confirmed some of thosedescribed for plants grown under low light, but export of ¹⁴Cby fed leaves was generally higher than reported for much ofthe earlier work. Lower leaves of young plants exported over50% of the ¹⁴C they fixed, although export fell sharply as theplants aged. Initially, the roots and apical tuft were strongsinks for assimilates, but they had declined in importance bythe time plants reached the nine-leaf stage. On the other hand,the stem became progressively more important as a sink for ¹⁴C-assimilates.Older, lower leaves exported more of their ¹⁴C-assimilates tothe upper part of the plant than to the roots, whereas youngleaves near the top of the plant exported more of their assimilatesto the roots. The stem internode immediately below a fed leafhad about twice the ¹⁴C activity of the internode above theleaf. Mature leaves above and below a fed leaf rarely importedmuch ¹⁴C, even when in the correct phyllotactic relationshipto the fed leaf. In the first 3 d after feeding leaf 5 of nine-leaf plants, theorganic and amino acid pools and the neutral fraction of theinternode below the fed leaf had most of the ¹⁴C activity, butby 49 d after feeding, the ethanolic-insoluble, starch and lipidfractions had most of the ¹⁴C activity. Glucose, fructose andsucrose were the main sugars in the stem. Although concentrationsof these sugars and starch declined in the stem as the plantsmatured, there was little evidence to indicate their use infruit production. Stems of plants defoliated at the 44-leafstage had lower concentrations of sugars and starch at maturity,and produced less fruit than the controls. It was concludedthat tomato is sink rather than source limited with respectto carbon assimilates, and that the storage of carbon in thestem for a long period is possibly a residual perennial traitin tomato.Copyright 1994, 1999 Academic Press Lycopersicon esculentum, tomato, assimilate distribution, ¹⁴C, internode storage, sink-source relationships, starch, stem reserves, sugars     

Pelagic food web processes in an oligotrophic lake

Major pelagic carbon pathways, including primary production, release of extracellular products (EOC), bacterial production and zooplankton grazing were measured in oligotrophic Lake Almind (Denmark) and in enclosures (7 m³) subjected to artificial eutrophication. Simultaneous measurements at three days interval of carbon exchange rates and pools allowed the construction of carbon flow scenarios over a nineteen day experimental period.The flow of organic carbon was dominated by phytoplankton EOC release, which amounted from 44 to 58% of the net fixation of inorganic carbon. Gross bacterial production accounted for 33 to 75% of the primary production. The lower values of EOC release (44%) and bacterial production (33%) were found in the enclosures with added nutrients. The release of recently fixed photosynthetic products was the most important source of organic carbon to the bacterioplankton. Uptake of dissolved free amino acids was responsible for 52 to 62% of the gross bacterial production. Thus, amino acids constituted a significant proportion of the EOC. Zooplankton (< 50 µm) grazing on algae and bacteria accounted only for a minor proportion of the particulate production in May. Circumstantial evidence is presented that suggests the chrysophycean alga Dinobryon was the most important bacterial remover.The results clearly demonstrated EOC release and bacterial metabolism to be key processes in pelagic carbon cycling in this oligotrophic lake.     

Metabolism of C1 unit precursors in Neurospora: Effects of media supplements on labelling of nucleic acids and protein

Mycclia of Neurospora crassa wild type (FE SC no. 853), harvestedduring the exponential phase of growth on defined minimal mediaincorporated glycine-2-¹⁴C, serine-3-¹⁴C and formate-¹⁴C intoproteins, DNA and RNA. Supplementing the growth medium with1 mM glycine increased the flow of glycine and formate carboninto these products. In contrast, this supplement decreasedthe incorporation of serine-¹⁴C. When such cultures were preincubatedfor 30 min with adenine, formaldehyde, formate or L-methionine,labelling of the nucleic acids and protein fractions by glycine-2-¹⁴Cwas altered. It is concluded that glycine increases the turnoverof C₁ units in Neurospora, resulting in greater contributionsof the C-2 in nucleic acid and protein synthesis. (Received May 14, 1977; )     

Effect of leaf age on light and dark 14CO2 fixation in a CAM plant, Bryophyllum calycinum

Leaves of different ages from B. calycinum were exposed to ¹⁴CO₂in light during day and night. The labelling pattern on thechromatogram differed with leaf age. Young leaves had similarpatterns to those of C3 plants during both day and night. Matureleaves showed high incorporation of ¹⁴C into C4 acids, especiallyat night. In contrast, no significant difference with leaf agewas observed in the pattern of dark ¹⁴CO₂ fixation products.Study of the enzyme activity and the content of titratable acidat each leaf age suggested that high incorporation of ¹⁴C inC4 acids during the night was due to the simultaneous absorptionof CO₂ by both enzymes RuDPcarboxylase and PEPcarboxylase. (Received November 24, 1977; )     

Glutamine synthesis in germinating castor bean endosperm

The pathway of glutamine synthesis in germinating castor beanendosperm was investigated by feeding experiments with (2,3-¹⁴C)succinateand by determining enzyme activities related to pyruvate formationand utilization. ¹⁴C of (2,3-¹⁴C)succinate was rapidly and sequentiallyincorporated into amino acids in the following order: aspartateor alanine, glutamate and glutamine. ¹⁴CO₂ was slowly released,especially during the early hours of incubation. Fluorocitrateinhibited ¹⁴CO₂ release while aminooxyacetate stimulated itslightly. Fluorocitrate inhibited the incorporation of ¹⁴C intoglutamate and glutamine. Aminooxyacetate inhibited ¹⁴C incorporationinto aspartate, alanine, glutamate and glutamine. Glutaminesynthetase activity was detected in a soluble fraction. NAD-malicenzyme activity was detected in mitochondria by sucrose densitygradient centrifugation. Activities of pyruvate decarboxylaseand aldehyde dehydrogenasewere detected. Aldehyde dehydrogenasewas partially purified about 60-fold by ammonium sulfate fractionationand the DEAE-cellulose chromatography. The Km values of theenzyme were 0.71 miu for NAD and 0.43 mM for acetaldehyde. Basedon these results and properties of pyruvate kinase reportedpreviously (9), the metabolism of pyruvate in cytosol and mitochondriawas discussed in connection with glutamine synthesis in germinatingcastor bean endosperm. (Received August 25, 1978; )