A simplified radioassay method of dihydropteroate synthetase activity in Escherichia coli and its application for an inhibition study of p-aminobenzoi acid derivatives

A method is described for the determination of the enzymic synthesis of 7,8-dihydropteroic acid in a cell-free system of E. coli, using ¹⁴C-labeled p-aminobenzoic acid as substrate. The p-aminobenzoic acid which is not incorporated into the product is extracted from the incubation mixture with ether at pH. 3.8. The radioactivity remaining in the water phase is a measure of the formed 7,8-dihydropteroic acid. This rapid procedure has been applied to the estimation of the inhibition indices of some competitive antagonists for the enzyme H₂-pteroate synthetase in E. coli.     

Liquid scintillation counting of 14C-labeled hemin and its application to the biosynthesis of heme in bone marrow

A rapid procedure is described for the liquid scintillation counting of ¹⁴C-labeled hemin isolated after incubation of bone marrow with radioactively labeled glycine-2-¹⁴C. The method has been applied for studies on the biosynthesis of heme in bone marrow of several animal species.     

The accumulation and metabolism of [C]hypoxanthine by slices of rabbit renal medulla

The uptake of hypoxanthine by rabbit renal medulla has been studied with in vitro conditions. Unlike the uptake by renal cortex slices reported earlier, no evidence was found for involvement of an organic cation transport system. Medullary accumulation of the ¹⁴C-labeled material occurred in the absence of O₂ if glucose was present as substrate. Uptake of the ¹⁴C label was not supported by other sugars or metabolic intermediates, however. Metabolic inhibitors reduced both aerobic and anaerobic uptake. Tissue extracts were subjected to high-voltage electrophoresis and gel filtration for identification of possible metabolites. These studies indicated that most of the uptake of ¹⁴C-labeled material was accounted for by its conversion to inosine-like and/or inosinic acid-like compounds. That is, when experimental conditions were designed to retard slice metabolism of hypoxanthine, tissue to medium ratios for ¹⁴C approximated 1.0. A third metabolite was found occasionally, but remains unidentified.     

Pancreas acinar cell differentiation. IV. Assay of nanogram levels of chymotrypsin by radioactively labeled synthetic substrate

A procedure for synthesizing ¹⁴C-labeled N-benzoyl-l-tyrosine ethyl ester with the label in the benzoyl group has been described. Using this substrate, which is specific for chymotrypsin, and employing trypsin activation of chymotrypsinogen in an incubation medium containing radiolabeled BTEE, we have presented a method which permits assay of nanogram quantities of chymotrypsin activity in organ-cultured tissue. The radiolabeled product of enzyme hydrolysis, N-benzoyltyrosine, is readily separated by paper chromatography from the unreacted labeled substrate and measured by radioactivity counting.     

Carbon dioxide fixation by detached cereal caryopses

Immature detached cereal caryopses from barley (Hordeum vulgare L. var distichum cv Midas) and wheat (Triticum aestivum L. cv Sicco) were shown to be capable of fixing externally supplied ¹⁴CO₂ in the light or dark. Green cross cells and the testa contained the majority of the ¹⁴C-labeled material. Some ¹⁴C-labeled material was also found in the outer, or transparent, layer and in the endosperm/embryo fraction. More ¹⁴C was recovered from caryopses when they were incubated in ¹⁴CO₂ without the transparent layer, thus suggesting that this layer is a barrier to the uptake of CO₂. In all cases, significant amounts of ¹⁴C-labeled material were found in caryopses after dark incubation with ¹⁴CO₂. Interestingly, CO₂ fixation in the chlorophyll-less mutant Albino lemma was significantly greater in the light than in the dark. The results indicate that intact caryopses have the ability to translocate ¹⁴C-labeled assimilate derived from external CO₂ to the endosperm/embryo. Carboxylating activity in the transparent layer appears to be confined to phosphoenolpyruvate carboxylase activity but that in the chloroplast-containing cross-cells may be accounted for by both ribulose-1,5-bisphosphate carboxylase-oxygenase and phosphoenolpyruvate carboxylase activity. Depending on a number of assumptions, the amount of CO₂ fixed is sufficient to account for about 2% of the weight of starch found in the mature caryopsis.     

Alteration of Acrylonitrile-Methylacrylate-Butadiene Terpolymer by Nocardia rhodochrous and Penicillium notatum

[¹⁴C]Barex-210, a terpolymer of acrylonitrile, methylacrylate, and butadiene, was tested for bioconversion. Powdered samples of polymer, each specifically ¹⁴C labeled at different carbon atoms of the polymer, were incubated with either Nocardia rhodochrous or Penicillium notatum in an enriched growth medium for various periods of time. After 6 months of incubation, the ¹⁴C-labeled polymer was transformed from a high-molecular-weight material completely soluble in dimethyl formamide (DMF) into both a lower-molecular-weight form still soluble in DMF and a second form that was no longer soluble in DMF. The amount of ¹⁴C-labeled carbon atoms converted into DMF-insoluble material was 8% of the backbone carbon-carbon atoms and 12% of the side-chain nitrile and acrylate atoms from the acrylonitrile-methylacrylate copolymer and 60% of the elastomer (acrylonitrile-butadiene copolymer) atoms. Metabolism of the polymer was not established from measurements of metabolic ¹⁴CO₂. Evolution of ¹⁴CO₂ amounted to only 0.3, 0.6, 1.8, and 3.3% of these four fractions, respectively. Although the transformation of high-molecular-weight polymer into DMF-insoluble material was rapid in the early stages of microbial growth, the accompanying CO₂ evolution was much slower. Further evidence of polymer alteration was indicated by the infrared spectrum of the insoluble material, which showed a disappearance of the nitrile and methylacrylate peaks.     

Functional characterization of single-chain factor X from rat liver

¹⁴C-Labeled single-chain factor X prepared by vitamin K-dependent carboxylation in vitro was partially purified by adsorption to BaSO₄ and chromatography on DEAE-Sephacel. Known activators of factor X were analyzed for their effect on the single-chain molecule. ¹⁴C-Labeled factor X antigens were recovered immunochemically from incubation mixtures and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation with trypsin resulted in the generation of factor Xa clotting activity, and the ¹⁴C-labeled product migrated after reduction with an apparent molecular weight of 22,500 ± 1500 (mean ± 1 SD). The light chain produced by factor Xa was similar to that produced by trypsin (M_r 24,500 ± 1500; mean ± 1 SD). Incubation of single-chain factor X with factor VII and thromboplastin, factor IXa, or the factor X activating enzyme from Russell's viper venom gave a reducible product with a light chain of higher apparent molecular weight (M_r 37,000–38,000). Incubation with factor VII and thromboplastin also resulted in the generation of factor Xa clotting activity. Incubation of single-chain factor X with platelets resulted in the binding of about 20% of the ¹⁴C. The bound ¹⁴C-labeled factor X antigen released by freezing and thawing in the presence of EDTA was reduced to give a ¹⁴C-labeled polypeptide with M_r 31,000. Walker 256 tumor cells bound about 30% of the ¹⁴C. The bound material, after reduction, gave a ¹⁴C-labeled polypeptide with M_r 23,000.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

A simple apparatus for the continuous monitoring of 14CO2 production from several small reaction mixtures

A simple apparatus is described which permits the continuous monitoring of ¹⁴CO₂ production from ten separate reaction mixtures simultaneously. The device is relatively simple and inexpensive to construct, makes use of small disposable incubation vials, and allows complete trapping of all ¹⁴CO₂ evolved in scintillation vials, where it can be easily counted. The use of this apparatus to determine the rates of metabolism by glomeruli of ¹⁴C-labeled substrates to ¹⁴CO₂ is described.     

THE DEPRESSION OF PHAGOCYTOSIS BY EXOGENOUS CYCLIC NUCLEOTIDES, PROSTAGLANDINS, AND THEOPHYLLINE

The effects of agents that elevate intracellular cyclic adenosine 3',5'-monophosphate (cAMP) have been studied with respect to phagocytosis by guinea pig polymorphonuclear leukocytes. The investigation depends upon the use of a precise method for following ingestion. Theophylline, dibutyryl cAMP, and prostaglandins inhibited the phagocytosis of starch particles. The inhibitions caused by prostaglandins E₁, E₂, and F_2α (PGE₁, PGE₂, and PGF_2α) were synergistic with that due to theophylline. Inhibition by PGA₁ and PGA₂ was not. At equal concentrations the order of increasing inhibition of phagocytosis (assayed at 10 min) by the prostaglandins was PGE₁ < PGF_2α < PGE₂ < PGA₁ = PGA₂. Our results are consistent with the hypothesis that increased intracellular levels of cAMP impair the phagocyte's ability to ingest particles. The mechanism of the inhibition has not been defined. The increment in oxidation of [1-¹⁴C]glucose to ¹⁴CO₂ that normally accompanies phagocytosis was found to be depressed in the presence of PGE₁ or theophylline, together or individually as expected from the inhibition of phagocytosis. Paradoxically, oxygen consumption although depressed by theophylline or PGE₁ plus theophylline, was stimulated by PGE₁ alone.     

A method for the analysis of tRNA patterns in Drosophila melanogaster using an amino acid analyser

Using tRNA and aminoacyl-tRNA synthetase preparations from Drosophila melanogaster, a method has been developed for simultaneously estimating levels of at least 15 different species of aminoacyl-tRNA. ¹⁴C-labeled aminoacyl-tRNA, which is formed during a single incubation of tRNA with a mixture of 15 ¹⁴C-labeled amino acids, is purified, hydrolysed, and the composition of the mixture of ¹⁴C-labeled amino acids so obtained is determined using an Amino Acid Analyser.The sensitivity of the method and the reproducibility of the results obtained are such that it is suitable for detecting changes in tRNA patterns in comparative studies.     

Microbial Decomposition of Synthetic 14C-Labeled Lignins in Nature: Lignin Biodegradation in a Variety of Natural Materials

Lignin biodegradation in a variety of natural materials was examined using specifically labeled synthetic ¹⁴C-lignins. Natural materials included soils, sediments, silage, steer bedding, and rumen contents. Both aerobic and anaerobic incubations were used. No ¹⁴C-labeled lignin biodegradation to labeled gaseous products under anaerobic conditions was observed. Aerobic ¹⁴C-labeled lignin mineralization varied with respect to type of natural material used, site, soil type and horizon, and temperature. The greatest observed degradation occurred in a soil from Yellowstone National Park and amounted to over 42% conversion of total radioactivity to ¹⁴CO₂ during 78 days of incubation. Amounts of ¹⁴C-labeled lignin mineralization in Wisconsin soils and sediments were significantly correlated with organic carbon, organic nitrogen, nitrate nitrogen, exchangeable calcium, and exchangeable potassium.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

In vitro synthesis of beta2-microglobulin by human fetal tissues.

Human fetal tissues were cultured in the presence of ¹⁴C-labeled amino acids and the culture fluids analyzed by radioimmunoelectrophoresis for the presence of radioactive β₂-microglobulin. Synthesis of the protein was demonstrated in all the tissues studied. Using the Ouchterlony method, β₂-microglobulin was also detected in culture media from serially transferred cell cultures of fetal tissues and established fetal cell strains.     

Parallelized small-scale production of uniformly C-labeled cell extract for quantitative metabolome analysis

The need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC–MS analysis. Therefore the application of uniformly (U) ¹³C-labeled cell extract as an internal standard has gained interest in recent years. In this study a multiple-step protocol has been developed for efficient preparation of U-¹³C-labeled Escherichia coli cell extracts in stirred-tank bioreactors on a milliliter scale with a minimal supply of costly ¹³C-labeled substrate. Significant reduction of fermentation medium salt concentration in the U-¹³C-labeled cell extract was achieved to reduce ion-suppression effects during mass-spectrometric analysis. Additionally, variation of reaction conditions in parallel-operated stirred-tank bioreactors on a milliliter scale enables the simultaneous preparation of U-¹³C-labeled cell extracts with varying metabolite concentrations, which is shown by an example of the labeled phosphoenolpyruvate level in E. coli.     

Degradation of softwood [14C lignin] lignocelluloses and its relation to the formation of humic substances in river and pond environments

The fate of lignin in water and sediment of the Garonne river (France) and of a pond in its floodplain was examined using specifically labeled [¹⁴C-lignin] lignocelluloses. No significant differences appeared in the mineralization rate of alder, poplar or willow [¹⁴C-lignin] in running water samples. Conversion of total radioactivity to ¹⁴CO₂ ranged between 18.7% and 24.4% after 120 days of incubation. Degree of ¹⁴C-labeled lignin mineralization in standing water and sediments was clearly lower, especially in submerged sediments, and was correlated with oxygen supply. After 60 days of incubation 3.3% to 7.9% of the ¹⁴C-labeled lignin was recovered in water samples as dissolved organic carbon originating from microbial metabolism. In water extracts from sediment the percentage of dissolved organic ¹⁴C was only 0.4% to 1.3% of the applied activity. In the humic fraction extracted from sediments it did not exceed 4.4% which was much lower than in soils. No significant difference appeared between river and pond conditions for humic substances formation.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

Determination of low levels of 14C radioactivity in cell exudates by means of ultraviolet photooxidation and a rapid 14CO2-collection technique

The low levels of ¹⁴C radioactivity found in many biological samples, such as cell exudates of marine algae, can be determined conveniently by uv photooxidation of the ¹⁴C-labeled compounds. The acid distillation of the resultant ¹⁴CO₂ and its rapid absorption (cf. 15 min) by a quaternary amine in a closed recirculating system prior to liquid scintillation counting provides a means of concentrating the ¹⁴C activity by up to 10 times. Quench problems are thus reduced to that of one compound, namely ¹⁴CO₂. The kinetics of photooxidation of various ¹⁴C-labeled compounds in seawater are complex but similar in form for the several different classes of compounds tested. The role of the nature and concentration of the oxidant, sample pH, and the source of uv irradiation during photooxidation are discussed.