High-voltage Paper Electrophoresis Research Articles

Production of Agrocinopine A by Ipomoea batatas Agrocinopine Synthase in Transgenic Tobacco and Its Effect on the Rhizosphere Microbial Community.

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation of plant vegetative tissues by pathogenic Agrobacterium spp., previous reports have indicated that T-DNA sequences originating from an ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) varieties analyzed. Expression of an Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by some strains of Agrobacterium spp. in crown gall. To validate the product synthesized by Ipomoea batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High-voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further mass spectrometry and nuclear magnetic resonance analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. A 16S ribosomal RNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria that can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Protocols for Glycosyltransferase Assays: Ganglioside Globoside and Lewis-X Intermediate-Lactosylceramide Biosyntheses in Eukaryotic Systems.

Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na2B4O7. Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer β1-4-galactosyltransferase), (Km of glucosylceramide=1.65×10-4M; Km for UDP-Gal=0.5×10-4M; V max is determined in the presence of optimum detergent concentrations (2-15mg/ml of Cutscum-Triton X-100, 2:1); Mn++ and Mg++, 10-20mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity.

Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin-lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose). Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed. Cress-seed exudate contained low-Mr, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µmLMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate. LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.

PP21 - The degradation of Vitamin C by reactive oxygen species

Vitamin C (ascorbate and dehydroascorbic acid) is vital for plants and found throughout the plant cell including in the apoplast. The structure of ascorbate was determined eighty years ago; however, many of its degradation pathways remain unclear. Numerous degradation products of ascorbate have been reported to occur in the apoplast but many still remain unidentified[1,2]. Ascorbate is well known as an antioxidant, and acts to quench reactive oxygen species (ROS), such as hydrogen peroxide and ozone in the plant apoplast. The immediate oxidation product of ascorbate is dehydroascorbic acid (DHA), which may be quickly hydrolysed to diketogulonic acid (DKG). The further reactions of radiolabelled and non-radiolabelled DHA and DKG with various ROS have been investigated. The resulting oxidation products were separated by high-voltage paper electrophoresis. Differences were observed in the products formed from the various ROS, allowing a unique fingerprint of oxidation products to be described for each ROS. Equally, different compounds were produced depending on the starting substrate; for example cyclic oxalyl threonate was only observed in the reactions of DHA and not DKG. Vitamin C is also a vital component of the human diet, and most dietary ascorbate comes from plants such as salads. The degradation of ascorbate during post-harvest processing and storage of salad leaves has been investigated. Studies are being conducted to determine the presence of ascorbate oxidation products within salad leaves. We thank the UK BBSRC and Vitacress Salads Ltd for funding.

Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Boron (B) is essential for plant cell-wall structure and membrane functions. Compared with its role in cross-linking the pectic domain rhamnogalacturonan II (RG-II), little information is known about the biological role of B in membranes. Here, we investigated the involvement of glycosylinositol phosphorylceramides (GIPCs), major components of lipid rafts, in the membrane requirement for B. Using thin-layer chromatography and mass spectrometry, we first characterized GIPCs from Rosa cell culture. The major GIPC has one hexose residue, one hexuronic acid residue, inositol phosphate, and a ceramide moiety with a C18 trihydroxylated mono-unsaturated long-chain base and a C24 monohydroxylated saturated fatty acid. Disrupting B bridging (by B starvation in vivo or by treatment with cold dilute HCl or with excess borate in vitro) enhanced the GIPCs’ extractability. As RG-II is the main B-binding site in plants, we investigated whether it could form a B-centred complex with GIPCs. Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC–B–RG-II complex. Last, using polyacrylamide gel electrophoresis, we showed that added GIPCs facilitate RG-II dimerization in vitro. We conclude that B plays a structural role in the plasma membrane. The disruption of membrane components by high borate may account for the phytotoxicity of excess B. Moreover, the in-vitro formation of a GIPC–B–RG-II complex gives the first molecular explanation of the wall–membrane attachment sites observed in vivo. Finally, our results suggest a role for GIPCs in the RG-II dimerization process.

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines.

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.

High-voltage paper electrophoresis (HVPE) of cell-wall building blocks and their metabolic precursors.

HVPE is an excellent and often overlooked method for obtaining objective and meaningful information about cell-wall "building blocks" and their metabolic precursors. It provides not only a means of analysis of known compounds but also an insight into the charge and/or mass of any unfamiliar compounds that may be encountered. It can be used preparatively or analytically. It can achieve either "class separations" (e.g. delivering all hexose monophosphates into a single pool) or the resolution of different compounds within a given class (e.g. ADP-Glc from UDP-Glc; or GlcA from GalA). All information from HVPE about charge and mass can be obtained on minute traces of analytes, especially those that have been radiolabelled, e.g. by in-vivo feeding of a (3)H- or (14)C-labelled precursor. HVPE does not usually damage the substance under investigation (unless staining is used), so samples of interest can be eluted intact from the paper ready for further analysis. Although HVPE is a technique that has been available for several decades, recently it has tended to be sidelined, possibly because the apparatus is not widely available. Interested scientists are invited to contact the author about the possibility of accessing the Edinburgh apparatus.

A Low Molecular Weight Proinflammatory Factor from Rat Spleen Lymphocytes. Isolation and Partial Characterization

A dialyzable low molecular weight proinflammatory factor (X (2)) from rat spleen lymphocytes was isolated through a combination of gel filtration and high voltage paper electrophoresis (HVE) and then partially characterized. It was able to potentiate the formation of carrageenin induced edema on the rat paw. Its amino acid analysis revealed Glu, Cys and Gly (1:1:1), but gammaGlu as N-terminal residue, initially suggesting oxidized glutathione (GSSG), since it showed exactly the same HV electrophoretic mobility as GSSG at pH 6.5. However, neither GSSG nor a synthetic homologue showed any proinflammatory activity. On basis of its infrared spectrum, HVE mobility and presence of a gamma-Glu-Cys-Gly (GSH) moiety, the hypothesis of identity of X (2) with leukotriene C(4) (LTC(4)) was raised. Once again it was not confirmed, since LTC(4) did not show any proinflammatory activity too, leading us to infer that, even excluding LTC(4), our data are consistent with a structure bearing a GSH moiety conjugated with a hydroxylated insaturated fatty acid chain which contributes a -COO(-) group, thus providing a final net charge of -2 at pH 6.5 and an Mr = 600-650.

Charge equilibria and p K values of 5-carboxypyranomalvidin-3-glucoside (vitisin A) by electrophoresis and absorption spectroscopy

Apparent p K values of the wine pigment, 5-carboxypyranomalvidin-3-glucoside (vitisin A), were determined using UV–vis spectroscopy, viz. p K a1=0.98 (±0.10), p K H1=4.51 (±0.03) and p K H2=7.57 (±0.02). An additional ionisation constant at high pH (p K a4=8.84±0.06) was established by high-voltage paper electrophoresis. These data in conjunction with previously published p K a values determined by high-voltage electrophoresis suggest that in wine (pH 3.2–3.8), 5-carboxypyranomalvidin-3-glucoside exists as a complex mixture of hydrated and non-hydrated, partially ionised species with the predominant species being the quinonoidal base ( λ max 498 nm).

Influence of structure on the ionisation constants of anthocyanin and anthocyanin-like wine pigments

The macro ionisation constants of malvidin-3-glucose, vitisin A and the two pairs of diastereoisomers of 8,8-ethyl-linked pigments formed between malvidin-3-glucose and catechin or epicatechin were determined using high voltage paper electrophoresis (HVPE) and compared as a function of their chemical structures. Their UV–vis spectral characteristics were also compared. The low p K a1 value of vitisin A (0.95 ± 0.10) compared to malvidin-3-glucose (1.76 ± 0.07) reflects the extended delocalisation of the former due to the presence of the fourth ring and the electron withdrawing effect of the carboxyl group. The presence of the ethyl bridge at the 8-position of the 8,8-ethyl-linked malvidin-3-glucose–(epi)catechin give rise to slightly higher p K a1 values (2.33–2.44) due to the electron donating effect of the alkyl group into the delocalised electron system. Similarly, the loss of a second proton, reflected by the p K a2 values is influenced by the 8-ethyl substituent. Small differences in p K a1 and p K a2 values between the different diasterioisomers reflects the impact of variation in three-dimensional structures. p K a1 and p K a2 values for these compounds determined by HPVE agree well with those determined by UV–vis spectroscopy indicating that the latter are true ionisation constants rather than a composite of ionisation and hydration constants.

Glycogen- and PP1c-targeting Subunit GM Is Phosphorylated at Ser48 by Sarcoplasmic Reticulum-bound Ca2+-Calmodulin Protein Kinase in Rabbit Fast Twitch Skeletal Muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).

Inducible Flavone in Oats (Avena sativa) Is a Novel Defense Against Plant-Parasitic Nematodes

ABSTRACT The induction of defense compounds in oats (Avena sativa) in response to invasion by parasitic nematodes and to application of the wound hormone methyl jasmonate was examined. Oats cv. Quoll seedlings were challenged with Pratylenchus neglectus, Heterodera avenae, and Ditylenchus dipsaci and treated with 1 x 10(-4) M methyl jasmonate. Three compounds, isolated in methanolic root and shoot extracts of oats, exhibiting an absorbance spectrum typical of flavone glycosides, were induced by nematode invasion and methyl jasmonate. These were identified as flavone-C-glycosides by mass spectrometry. The effect of the flavone-C-glycosides on the invasion by and development of cereal cyst nematode H. avenae was assessed using methanolic extracts of shoots and roots from methyl jasmonate-treated plants. Both extracts impaired nematode invasion and development. When the extracts were fractionated by high voltage paper electrophoresis, only one flavone-C-glycoside, O-methyl-apigenin-C-deoxyhexoside-O-hexoside, inhibited nematode invasion. The protective effect of the induction of flavone-C-glycosides in oats by methyl jasmonate was evaluated against H. avenae and P. neglectus. Treatment with methyl jasmonate reduced invasion of both nematodes and increased plant mass, compensating for damage caused by the nematodes, and is attributed to the active flavone-C-glycoside. The active compound, O-methyl-apigenin-C-deoxyhexoside-O-hexoside, has not been implicated previously in plant defense against any pest or pathogen, and appears to provide protection against the major cereal nematodes Heterodera and Pratylenchus.

Formation of S-[2-carboxy-1-(1H-imidazol-4-yl) ethyl]glutathione, a new metabolite of L-histidine, from cis-urocanic acid and glutathione by the action of glutathione S-transferase.

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

Purification of anthocyanins from species of Banksia and Acacia using high-voltage paper electrophoresis.

A new method has been developed for the isolation and rapid identification of anthocyanins from two floricultural crops based on the use of high-voltage paper electrophoresis with bisulphite buffer. Using this method, anthocyanin pigments were successfully purified as their negatively charged bisulphite-addition compounds from crude extracts of plant tissue. In conjunction with liquid chromatography-electrospray mass spectrometry, the method enabled the anthocyanins from the flowers of two Banksia species and the leaves of two Acacia species to be identified. The Banksia flowers contained both cyanidin and peonidin-based pigments, while the Acacia leaves contained cyanidin and delphinidin derivatives.

Determination of S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione, a novel metabolite of L-histidine, in tissue extracts from sunlight-irradiated rat by capillary electrophoresis.

Exposure of the skin to sunlight results in an increase in the content of epidermal urocanic acid, a key metabolite of L-histidine, and some portions of the metabolite penetrate into the body fluid. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of glutathione and urocanic acid, was proposed to be an origin of a urinary compound, S-[2-carboxy-1-(1 H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). Various catabolites of Cys(CIE) were also isolated from human urine previously. However, no direct evidence to show the existence of GS(CIE) as a biological material had been found. By using capillary electrophoresis, the glutathione adduct has now been found in the extracts of rat tissues from the kidney, liver, skin and blood when the rat was kept under conditions of sunlight irradiation after the fur on the dorsal skin had been clipped. On the other hand, no or a trace of GS(CIE) was determined in rat tissue extracts when the animal was kept indoor in usual manner. The glutathione adduct was isolated from the kidney extract of the sunlight-irradiated rat using ion-exchangers and high-voltage paper electrophoresis, and determined by fast-atom-bombardment mass spectrometry. These results indicate that GS(CIE) formation actually occurs in the body and that the formation is accelerated by exposing the rat to sunlight irradiation. From these findings, we propose an alternative pathway of histidine metabolism which is initiated by the adduction of urocanic acid to glutathione to form GS(CIE) and terminates with the formation of the urinary compounds via Cys(CIE).

The interactions of 54Mn with humic acids in freshwater systems

The interactions of 54Mn radionuclide with humic acids (HA) isolated from the sediment of a tropical estuary at Mahakam were studied in freshwater system from the Butoniga impoundment by high voltage paper electrophoresis. This way, it was possible to detect cationic, anionic and immobile 54Mn species, and to calculate their electrophoretic mobilities. In freshwater from the Butoniga impoundment in the pH range from 8.1 to 10.5 on average 69% of 54Mn is found in the cationic zone, which corresponds to the mobility of Mn(H 2O) 6 2+ ions. The immobile electrophoretic zone of 54Mn present in the freshwater at pH>10 in the absence of HA probably consists of the sparingly soluble or precipitated hydrolytic 54Mn compounds. These immobile 54Mn forms are solubilized after the addition of HA at elevated pH values. This effect is pronounced by the increased concentration of HA and the pH, especially at HA concentration of 200 mg l −1 and pH 11.0. The cationic forms of 54Mn-HA present have lower electrophoretic mobilities [ u −=(−1.16±0.05)×10 −4 cm 2 V −1 s −1 at pH 10.5 and u −=(−1.50±0.27)×10 −4 cm 2 V −1 s −1 at pH 11.0] than the cationic forms of 54Mn in freshwater system without HA added [ u −=(−3.09±0.16)×10 −4 cm 2 V −1 s −1 at pH 10.5 and u −=(−2.90±0.26)×10 −4 cm 2 V −1 s −1 at pH 11.0] indicating the formation of new 54Mn cationic forms after the interaction with HA.

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.

The interactions of 54Mn with humic substances of freshwater sediment origin

The interactions of 54Mn radionuclide with humic (HAB) and fulvic acid (FAB) isolated from freshwater sediment of the Butoniga impoundment were studied in a freshwater system from Butoniga impoundment, and in a diluted seawater system from the Rašla Bay at elevated pH by high-voltage paper electrophoresis. In this way, it was possible to detect cationic, anionic and immobile 54Mn species, and to calculate their electrophoretic mobilities. The interaction of 54 Mn with humic substances (HS) in a freshwater system is demonstrated by the formation of an electrophoretically immobile zone and the formation of the cationic forms of 54Mn having smaller electrophoretic mobilities than cationic forms of 54Mn in a freshwater system without HS added. The amount of cationic forms decreases in the presence of HS. This effect is enhanced with an increase in HS concentration in the range 5–100 mg l −1 and in an increase in pH from 9.5 to 10.5. The addition of HAB up to 100 mg l −1 and the increase of pH from 7.9 to 9.0 in diluted seawater systems do not cause significant changes of cationic electrophoretic mobilities of 54Mn, nor the formation of an electrophoretically immobile form of 54Mn.

Isolation and characterization of [formula omitted], a new metabolite of histidine, from normal human urine and its formation from [formula omitted

N- Acetyl-S-[2- carboxy-1-(1 H- imidazol-4- yl)ethyl]- l-cysteine ( I), a new imidazole compound with a sulfur-containing side chain, was isolated from normal human urine by ion-exchange column chromatography, and characterized by physicochemical analyses involving 1H-NMR spectrometry, mass spectrometry and high-voltage paper electrophoresis as well as chemical synthesis. Approximately five milligrams of crystals of the compound were obtained from 450 litres of the urine. Compound I was synthesized by the addition of N- acetyl- l-cysteine to urocanic acid. The compound was also formed by incubation of S-[2- carboxy-1-(1 H- imidazol-4- yl)ethyl]- l-cysteine ( II) with acetyl-CoA in the use of rat kidney or liver homogenate as an enzyme source in a Tris buffer at pH 7.4. Rat brain and spleen homogenates were the less or no effective preparations as the enzyme source. On the other hand, little N-acetylation of a diastereomer of compound II occurred in enzymatic reactions with rat tissue homogenates. Compound I was degraded to compound II by rat kidney or liver homogenate. These results suggest that compound I is a new N-acetylated metabolite of compound II, a compound previously found in human urine, and that the acetylating enzyme recognizes stereoisomerism of asymmetric carbon atoms on the molecule of compound II. These findings support an alternative pathway of l-histidine catabolism initiated by the adduction of glutathione and/or cysteine to urocanic acid, the first catabolite of histidine.

The interactions of 54Mn with aminopolycarboxylic acids in aquatic systems

The interactions of 54Mn radionuclide with ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) were studied in freshwater system (Butoniga impoundment), and in diluted seawater system (the Raša Bay) by high voltage paper electrophoresis. Depending on the EDTA or NTA concentrations it was possible to detect the distribution of cationic, anionic and immobile 54Mn species, and to calculate their electrophoretic mobilities. The apparent concentration stability constants of 54Mn-EDTA or 54Mn-NTA complexes were calculated from the respective titration curves. Logarithmic values of the apparent concentration stability constants for 54Mn-EDTA complex corrected for the influence of pH, K c( αH) , are in the range 6.20–7.38 in two different water types, while corrected for the effect of pH and Ca 2+, K c(αL), in 5% seawater amount from 11.29 to 11.89. Logarithmic values of the apparent concentration stability constants for 54Mn-NTA complex corrected for the influence of pH, K c( αH) , are in the range 6.42-5.21 in two different water types, while corrected for the effect of pH and Ca 2+, K c( αL) , in 5% seawater amount from 6.92 to 6.47.