The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Auxin transport in Avena: I. Indoleacetic Acid-C distributions and speeds

Measurements have been made on the initial stages of the transport of carbon-14-labeled indoleacetic acid in the coleoptile of Avena sativa L. Concentrations of mobile and immobilized indoleacetic acid are related to both distance and time during the first 2 hours after application of the indoleacetic acid at several concentrations to the top of the decapitated coleoptile.     

Hormonal Relations in the Phototropic Response: III. The Movement of C-labeled and Endogenous Indoleacetic Acid in Phototropically Stimulated Zea Coleoptiles

The effect of phototropic stimulation of Zea coleoptile tips on the distribution of both endogenous indoleacetic acid (IAA) and applied C¹⁴-labeled IAA was determined. The tips rested on bisected agar blocks. More IAA was found in the blocks under the shaded side of the coleoptile tips than those under the irradiated side. However, no significant difference was observed between the total amounts of IAA, endogenous or labeled, in the irradiated and shaded sides of the experimental system. In addition, less endogenous auxin was found in the shaded tissues than in their irradiated counterparts. It is suggested that phototropism following unilateral irradiation with first positive radiant densities might be a consequence of lateral inequalities in the ability of the irradiated and shaded tissues to transport auxin basipetally.     

Oxidative metabolism of propionate in Rhodospirillum rubrum

Summary Studies were conducted of the oxidative metabolism of propionate in Rhodospirillum rubrum, using C¹⁴-labeled compounds. The results, taken in conjunction with a carbon dioxide requirement described previously (Clayton et al., 1957), reveal that the first step is a carboxylation of propionic acid, yielding succinic acid. This result is confirmed through manometric studies of inhibition by malonate.     

Changes in the pattern of protein synthesis induced by 3-indolylacetic Acid

Experiments have been performed to investigate whether indoleacetic acid changes the balance between the rates of synthesis of different kinds of proteins. Sub-apical sections of etiolated peas were incubated with ¹⁴C- or ³H-labeled amino acid, and combined to give dual-labeled tissue. Cell fractions were prepared by differential centrifugation, and the dual-labeled protein of each fraction analyzed by gel-filtration. When 2 × 10⁻⁵ m indoleacetic acid was included with ¹⁴C-labeled amino acid, but not with the ³H-labeled amino acid, pronounced changes occurred in the pattern of incorporation of the ¹⁴C label into protein. These changes were greatest in the proteins of the particulate fraction which included nuclear material. Although the pattern of incorporation of lysine was shown to be different from that of leucine, the changes induced by indoleacetic acid were quantitatively similar whichever amino acid was used as a precursor. Dual-labeled protein was further fractionated using column chromatography on DEAE-cellulose. The results suggested that the effect of indoleacetic acid may not be completely general, and that the pattern of synthesis of many proteins may be unaltered by indoleacetic acid. When tissue was preincubated with 10 μg/ml actinomycin D for 30 minutes, incorporation of amino acid into protein was reduced but not abolished. Actinomycin D did, however, prevent the changes in the pattern of protein synthesis which were induced by indoleacetic acid.     

TRANSLOCATION IN MACROCYSTIS. III. COMPOSITION OF SIEVE TUBE EXUDATE AND IDENTIFICATION OF THE MAJOR C14-LABELED PRODUCTS1

The major C¹⁴-labeled substance in sieve tube exudate of M. pyrifera is D-mannitol, comprising 3.6% (w/v). No sugars are detectable. Certain amino acids also possess some C¹⁴-labeling and occur in significantly high concentrations in exudate. The exudate contains negligible ether-soluble lipid, but has a large amount of protein and a high concentration of K⁺ Neither protein nor lipid become labeled significantly in sieve tubes during short-term translocation experiments with C¹⁴. In general the chemical composition of the assimilate stream is comparable to that of vascular plants and does not, consequently, necessitate a different mechanism for translocation.     

Comparison of Protein Precipitants for the Determination of Free Amino Acids in Plasma

The present study was undertaken to select the most preferred deproteinizing agent for microbiological or chromatographic determination of free amino acids in plasma. The highest quantities of amino acids were obtained from samples treated with picric acid, ethanol or sulfosalicylic acid. It was also found from examining the recoveries of ¹⁴C-labeled amino acids added to plasma following treatment with picric acid, ethanol and sulfosalicylic acid that one kind of deproteinizing agent is not equally effective with all amino acids. Picric acid treatment gave the most reliable results for determination of amino acids except the basic amino acids, alanine and tryptophan. Sulfosalicylic acid was the most recommended deproteinizing agent for basic amino acids and ethanol was good for the assay of alanine and tryptophan. The response of d-amino acids to the deproteinizing agents tested was not always similar to that of their l-isomers.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Conversion of indole-3-ethanol to indole-3-acetic Acid in cucumber seedling shoots

Indoleethanol-¹⁴C was applied to intact cucumber seedlings and to hypocotyl segments. The presence of indoleacetic acid-¹⁴C in tissue extracts was demonstrated by thin layer radiochromatography. There was no evidence of conversion of indoleacetic acid to indoleethanol. It is suggested that the growth-promoting activity of indoleethanol is due to its conversion to indoleacetic acid.     

Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Increased biooxidation of lignosulfonates by Sporotrichum pulverulentum in the presence of polyacrylic acid

Summary The superiority of polyacrylic acid used as a buffer at 0.25% (w/v) during biodegradation of high molecular weight ¹⁴C-labeled lignosulfonates (LS) by the white rot fungus Sporotrichum pulverulentum is demonstrated. Compared to 2,2-dimethylsuccinate (DMS) the release of ¹⁴CO₂ from the LS occurs earlier, does not show the levelling-off symptom and reaches higher levels. Changes in pH values of the medium cannot be correlated with the stimulating effects of polyacrylic acid on the ligninolytic activity of the fungus. It seems that interaction between LS and also of dehydropolymers of coniferyl alcohol (DHP) with the polymeric buffer increases the accessibility to the fungus.     

TRANSLOCATION IN MACROCYSTIS. III. COMPOSITION OF SIEVE TUBE EXUDATE AND IDENTIFICATION OF THE MAJOR C14-LABELED PRODUCTS1

The major C¹⁴-labeled substance in sieve tube exudate of M. pyrifera is D-mannitol, comprising 3.6% (w/v). No sugars are detectable. Certain amino acids also possess some CWabeling and occur in significantly high concentrations in exudate. The exudate contains negligible ether-soluble lipid, but has a large amount of protein and a high concentration of K⁺. Neither protein nor lipid become labeled significantly in sieve tubes during short-term translocation experiments with C¹⁴. In general the chemical composition of the assimilate stream is comparable to that of vascular plants and does not, consequently, necessitate a different mechanism for translocation.     

Biosynthesis of radioactive sugars using cantaloupe leaves

Cantaloupe leaves having a high initial starch content were employed for the preparation of C¹⁴-labeled glucose by photosynthesis. The leaves were used without the customary carbohydrate-depletion procedure. The starch was extracted with perchloric acid. The labeled glucose obtained by the hydrolysis of the starch must be carefully crystallized in order to eliminate radioactive impurities.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid     

Correlative inhibition of lateral bud growth in Phaseolus vulgaris L. timing of bud growth following decapitation

Summary On intact, 3-week-old plants of Phaseolus the larger bud in the axils of the primary leaves shows slow, continuous elongation growth. Release from correlative inhibition can be detected within 30 min following decapitation. When 0.1% indoleacetic acid in lanolin is applied to the decapitated stem stump, the lateral bud shows slow growth during the first 7 h, then stops completely for a further 15 h but after 2 days a further gradual increase in length is observed.The movement of ¹⁴C-labelled assimilates from the subtending primary leaf into the lateral bud increases following removal of the shoot apex. When indole acetic acid is applied to decapitated plants the ability of the buds to import ¹⁴C increases for 5–7 h and then declines to a negligible amount. Little or no radioactivity from tritiated indoleacetic acid is transported into the lateral buds of decapitated plants during the first 48 h following removal of the apex and it appears that rapid metabolism of the compound occurs in the stem tissues.     

Inhibition of Lettuce Seed Germination and Root Elongation by Derivatives of Auxin and Reversal by Derivatives of Cycocel

Lettuce seed germination or lettuce root elongation after germination in water was inhibited by 5.7 × 10^-6M indoleacetic acid (IAA) and designated other IAA derivatives. These IAA-inhibited growth responses were reversed by 10^-5 to 10^-6M Cycocel, (2-chloroethyl) trimethylammonium chloride, which alone was without effect. Only those Cycocel analogs, which have previously been shown to be active as plant growth retardants, were effective in reversing IAA inhibition of germination or root elongation. These results are consistent with the concept that Cycocel at low concentrations acts as an auxin antagonist. However. Cycocel did not reverse the inhibitory effects from indole-3-propionic acid or indole-3-butyric acid.     

C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of ¹⁴C₂H₄ into the tissue and (b) the conversion of ¹⁴C₂H₄ to ¹⁴CO₂, was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to ¹⁴C₂H₄ markedly reduced both a and b. Increasing the ¹⁴C₂H₄ concentration from 0.14 to over 100 μl/l progressively increased the rate of a and b with tissue incorporation being greater than ¹⁴C₂H₄ to ¹⁴CO₂ conversion only below 0.3 μl/l ¹⁴C₂H₄. Reduction of the O₂ concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO₂ (5%) severely inhibited ¹⁴C₂H₄ to ¹⁴CO₂ conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during ¹⁴C₂H₄ treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.     

Plasma membrane ectoglycosyltransferase activity of L1210 murine leukemic cells

L1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N-acetylneuraminic acid-[C¹⁴] than control cells when incubated for 30 minutes with cytidine 5′-monophosphate N-acetylneuraminic-[C¹⁴] acid and three times more galactose-[C¹⁴] when incubated with uridine diphosphate galactose-[C¹⁴]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N-acetylneuraminic acid- [C¹⁴] could be removed by further treatment of these cells with neuraminidase. The incorporation of N-acetylneuraminic acid- [C¹⁴] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba⁺⁺ and Ca⁺⁺ ions at 8.3 mM increased the incorporation of N-acetylneuraminic acid- [C¹⁴] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn⁺⁺ or Hg⁺⁺ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N-acetylneuraminic acid- [C¹⁴] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring