Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

Preparation of radioactive gibberellin a(1) and its metabolism in dwarf peas

Gibberellin A₁-3,4-³H was prepared by selective catalytic reduction of gibberellic acid with a mixture of tritium and hydrogen. ³H-GA₁ was applied at physiological concentrations to dwarf peas and the metabolism of the hormone was investigated. ³H-GA₁ was converted to an acidic, biologically active compound. Radioactive but biologically inactive compounds were also found in the neutral fraction and could not be converted to acidic gibberellins by hydrolysis. No attachment of gibberellin to any macromolecular fraction was evident.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

Isolation of gibberellin precursors from heavily pigmented tissues

The kauranoid precursors of gibberellins are difficult to isolate from heavily pigmented plant tissues. In this paper, we describe relatively simple and efficient procedures for the purification of these compounds from tissues containing chlorophyll and other high molecular weight pigments. Extracts of shoots from Thlaspi arvense L. were subjected first to size exclusion chromatography using ethyl acetate as the eluting solvent. This procedure resulted in the separation of kauranoids as a class of compounds from chlorophyll. Typically, a 90% reduction in mass of the kauranoid enriched-fraction was observed. This fraction was subjected to reverse phase high performance liquid chromatography and individual fractions analyzed by combined gas chromatography-mass spectrometry. Five kauranoids were identified in shoot extracts of T. arvense: ent-kaur-16-ene, ent-kaur-16-en-19-ol, ent-kaur-16-en-19-oic acid, trachylobanoic acid, and 7β, 13-dihydroxykaurenolide. The metabolic relationships of these compounds to the gibberellins previously identified in this species (JD Metzger, MC Mardaus [1986] Plant Physiol 80: 396-402) are discussed. In addition, the utility of size exclusion chromatography in preparative situations is demonstrated by the purification of ent-kaurenoic acid in milligram quantities from the florets of Helianthus annuus L.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

The metabolism of organic acids by a marine pennate diatom

Cocconeis diminuta, a marine benthic diatom, metabolizes acetate and lactate-¹⁴C. In the light, the major product was lipid, whereas in the dark, CO₂ was the major product. Analysis of proteins synthesized in the presence of acetate or lactate showed that radioactivity was incorporated predominantly into the glutamate family of amino acids and those amino acids related directly to the substrate. Light and dark assimilation of substrate was inhibited slightly by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea and 2,4-dinitrophenol. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea caused a pattern of metabolism of acetate in the light characteristic of that which occurs in the dark. Monofluoroacetic acid inhibited assimilation considerably in the dark, but less in the light. The level of enzymes of the tricarboxylic acid cycle and NADH-oxidase were found to be about the same as those in other autotrophs. The metabolism of acetate and lactate is discussed in relation to the autotrophic mode of nutrition of Cocconeis diminuta.     

Metabolism of Acetate, Methanol, and Methylated Amines in Intertidal Sediments of Lowes Cove, Maine

The fates and the rates of metabolism of acetate, trimethylamine, methylamine, and methanol were examined to determine the significance of these compounds as in situ methane precursors in surface sediments of an intertidal zone in Maine. Concentrations of these potential methane precursors were generally <3 μM, with the exception of sediments containing fragments of the seaweed Ascophyllum nodosum, in which acetate was 96 μM. [2-¹⁴C]acetate turnover in all samples was rapid (turnover time <2 h), with ¹⁴CO₂ as the primary product. [¹⁴C]trimethylamine and methylamine turnover times were slower (>8 h) and were characterized by formation of both ¹⁴CH₄ and ¹⁴CO₂. Ratios of ¹⁴CH₄/¹⁴CO₂ from [¹⁴C]trimethylamine and methylamine in uninhibited sediments indicated that a significant fraction of these substrates were catabolized via a non-methanogenic process. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [¹⁴C]methanol was oxidized relatively slowly compared with the other substrates and was catabolized mainly to ¹⁴CO₂. Results from experiments with molybdate and 2-bromoethanesulfonic acid suggested that methanol was oxidized primarily through sulfate reduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1% of total methane production.     

Rapid Identification of α-Glucosidase Inhibitors from Phlomis tuberosa by Sepbox Chromatography and Thin-Layer Chromatography Bioautography

Alpha-glucosidase inhibitors currently form an important basis for developing novel drugs for diabetes treatment. In our preliminary tests, the ethyl acetate fraction of Phlomis tuberosa extracts showed significant α-glucosidase inhibitory activity (IC₅₀ = 100 μg/mL). In the present study, a combined method using Sepbox chromatography and thin-layer chromatography (TLC) bioautography was developed to probe α-glucosidase inhibitors further. The ethyl acetate fraction of P. tuberosa extracts was separated into 150 individual subfractions within 20 h using Sepbox chromatography. Then, under the guidance of TLC bioautography, 20 compounds were successfully isolated from these fractions, including four new diterpenoids [14-hydroxyabieta-8,11,13-triene-11-carbaldehyde-18-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (1), 14-hydroxyabieta-8,11,13-triene-17-oic-12-carboxy-13-(1-hydroxy-1-methylethyl)-lactone (2), 14,16-dihydroxyabieta-8,11,13-triene-15,17-dioic acid (3), and phlomisol (15,16-eposy-8,13(16),14-labdatrien-19-ol) (4)], and 16 known compounds. Activity estimation indicated that 15 compounds showed more potent α-glucosidase inhibitory effects (with IC₅₀ values in the range 0.067–1.203 mM) than the positive control, acarbose (IC₅₀ = 3.72 ± 0.113 mM). This is the first report of separation of α-glucosidase inhibitors from P. tuberosa.     

Gibberellins in the embryonic axes of tall and dwarf beans and their changes with initial growth

Ten gibberellin-like activities were detected in the dry embryonicaxes of tall (cv. Kentucky Wonder) and dwarf (cv. Masterpiece)beans (Phaseolus vulgaris L.) using the lettuce hypocotyl assayof thin-layer chromatograms; 2 in the non-acidic ethyl acetatefraction (NEI, NEII), 3 in the acidic ethyl acetate fraction(AEI, AEII, AEIII), 2 in the non-acidic n-butanol fraction (NBI,NBII) and 3 in the acidic n-butanol fraction (ABI, ABII, ABIII).There was no qualitative difference in these gibberellins betweenthe tall and dwarf axes, but all, particularly AEIII, NBII andABIII as the main gibberellins in the axes, were contained muchmore abundantly in the tall axes. In both axes the gibberellinactivities of most fractions decreased during germination.Theamounts of some gibberellins in tall axes without cotyledonswere greater than those in axes with cotyledons at 48–72hr of germination. Neither AMO-1618 nor CCC caused significantreduction in the levels of the gibberellins. Axis growth inthe early germinating period depended on the gibberellins storedin the axis, itself. (Received November 26, 1974; )     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.     

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.     

The immunoassay of gibberellins

The development of sensitive and specific solid-phase enzyme immunoassays for gibberellic acid and gibberellins A₄ and A₇ is reported. The use of antisera of high apparent affinity (K_a over 10¹⁰ l mol^-1) in conjunction with alkaline phosphatase-labeled gibberellins allows, with minimum procedural effort, the quantitative determination of sub-picogram amounts of these gibberellins. The assays reported here are applicable to most gibberellins and can be set up with 1–1.5 mg of starting material. They represent the most sensitive methods for gibberellin determination known.Abbreviations GA gibberellin - GA₃ gibberellic acid - TLC thin-layer-chromatography     

Stimulation of h efflux and inhibition of photosynthesis by esters of carboxylic acids

Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H⁺ efflux in the dark or light and photosynthetic O₂ production. Addition of 0.15 to 1.5 millimolar malathion, α-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H⁺ efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of α-naphthylacetate stimulated the normal rate of H⁺ efflux, 0.77 nanomoles H⁺ per 10⁶ cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H⁺ efflux after the normal rate of H⁺ efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N₂. Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, α-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, α-naphthol, and p-nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H⁺ efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes.     

Gibberellin-sensitive Suspension Cultures

Suspension cultures were incubated in the presence and absence of gibberellic acid (GA₃) in an attempt to define a new experimental system for study of the molecular action of gibberellins upon growth. Unlike many suspension cultures, an auxin-independent green clone from spinach (Spinacia oleracea L.) and an auxin-dependent line of “Paul's Scarlet” rose (Rosa sp.) were promoted in expansion growth by GA₃ at 10⁻¹¹ to 10⁻⁶ molar. In Rosa the cells also elongated upon GA₃ treatment whereas in Spinacia they remained isodiametric.     

Microbial Assimilation of Hydrocarbons: Identification of Phospholipids

The distribution of phospholipids derived from Micrococcus cerificans was determined under a variety of nutritive conditions. Cells were grown with hexadecane, heptadecane, or acetate serving as the sole carbon source. Total lipid was isolated by chloroform-methanol extraction, and the phospholipid fraction was isolated by silicic acid column chromatography. The phospholipids were characterized by silicic acid chromatography, by thin-layer chromatography, and by identification of water-soluble products resulting from acid hydrolysis of purified phospholipids. Major phospholipids characterized were phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Minor phospholipids were phosphatidylglycerol phosphate and phosphatidylserine. Trace amounts of methylated derivatives of phosphatidylethanolamine were determined by incorporation of ¹⁴C from ¹⁴C-methylmethionine. These experiments demonstrated the presence of phosphatidyl-N-methylethanolamine, phosphatidyl-N,N′-dimethylethanolamine, and phosphatidylcholine in trace quantities. Pulse labeling with ¹⁴C-serine demonstrated the direct incorporation of serine into phosphatidylserine followed by decarboxylation to phosphatidylethanolamine.     

5-Hydroxytryptamine metabolism by the nuclear fraction of rat-liver homogenate

1. The metabolism of 5-hydroxy[1′-¹⁴C]tryptamine creatinine sulphate in the nuclear fraction of rat-liver homogenate was studied. In the incubation mixture five metabolites were found. 2. Two metabolites were not radioactive; one of them was identified as 5-hydroxyindole-3-carboxylic acid and the second tentatively as 5-hydroxyindole-3-aldehyde. 3. 5-Hydroxyindol-3-ylacetic acid, 1′-N-acetyl-5-hydroxytryptamine and 5-hydroxytryptophol were not precursors of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid. 4. It was shown that the metabolism of 5-hydroxytryptamine in the nuclear fraction involves monoamine oxidase, the precursor of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid being most probably 5-hydroxyindol-3-ylacetaldehyde.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates

The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO₂ under acidic conditions, N⁵-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N²,N²-dimethylguanosine generated no product. In a similar experiment without NO₂^–, participation of a Dimroth rearrangement was ruled out. In the guanosine–HNO₂ reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20°C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by HNO₂ is proposed.     

Fermentative Metabolism of Chlamydomonas reinhardii: III. Photoassimilation of Acetate

The anaerobic photodissimilation of acetate by Chlamydomonas reinhardii F-60 adapted to a hydrogen metabolism was studied utilizing manometric and isotopic techniques. The rate of photoanaerobic (N₂) acetate uptake was approximately 20 μmoles per milligram chlorophyll per hour or one-half that of the photoaerobic (air) rate. Under N₂, cells produced 1.7 moles H₂ and 0.8 mole CO₂ per mole of acetate consumed. Gas production and acetate uptake were inhibited by monofluoroacetic acid (MFA), 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and by H₂. Acetate uptake was inhibited about 50% by 5% H₂ (95% N₂). H₂ in the presence of MFA or DCMU stimulated acetate uptake and the result was interpreted to indicate a transition from oxidative to reductive metabolism. Carbon-14 from both [1-¹⁴C]- and [2-¹⁴C]acetate was incorporated under N₂ or H₂ into CO₂, lipids, and carbohydrates. The methyl carbon of acetate accumulated principally (75-80%) in the lipid and carbohydrate fractions, whereas the carboxyl carbon contributed isotope primarily to CO₂ (56%) in N₂. The presence of H₂ caused a decrease in carbon lost from the cell as CO₂ and a greater proportion of the acetate was incorporated into lipid. The results support the occurrence of anaerobic and light-dependent citric acid and glyoxylate cycles which affect the conversion of acetate to CO₂ and H₂ prior to its conversion to cellular material.     

Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.