Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s

Geometrical configuration of the polyene chain of approximately 40 mono- and di-cis carotenoids was determined from 1970 through 1990. Subsequently, the kinetic, equilibrium and thermodynamic parameters (k, K, A, E_A, ΔH^#, ΔG^#, ΔS^#) of the reversible thermal isomerization of several symmetrical and unsymmetrical carotenoids were calculated. The rate of the iodine-catalyzed photoisomerization of (all-E)-, (9Z)- and (13Z)-zeaxanthin was compared and the ‘specific rate’ (per unit light energy at given wavelengths) of the iodine-catalyzed photoisomerization for several (13Z)-carotenoids was investigated. As the missing links of the biosynthetic pathway of paprika-carotenoids, carotenoids containing new end groups were isolated; their sterically unhindered mono-cis isomers were also prepared and their geometrical configuration was determined. The investigation concentrated on the substrate specificity of the enzyme violaxanthin-deepoxidase, the light-induced formation of (13Z)-violaxanthin in green leaves, the binding of xanthophylls to the bulk light-harvesting complex (LHC) of photosystem II in higher plants, the biochemical basis of color as an aesthetic quality in Citrus-fruits and the (9Z)-epoxycarotenoid cleavage enzyme activity for ABA biosynthesis. Recently (9Z)-capsanthin-5,6-epoxide and capsoneoxanthin, two novel carotenoids have been isolated from natural sources.     

Independent blood-brain barrier transport systems for nucleic acid precursors

The blood-brain barrier permeability to certain ¹⁴C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15 s later. The amount of ¹⁴C-labelled base or nucleoside remaining in brain was expressed in relation to ³H₂O (a highly diffusible internal standard) and ^113mIn-labelled EDTA (an essentially non-diffusible internal standard).Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine.Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (K_m = 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (K_m = 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [¹⁴C]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps.The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD⁺, cyclic AMP, NADP⁺ and uridinediphosphoglucose (UDPG).In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.     

Synthesis of diorganotin(IV) complexes with nucleosides and their Characterization by Mössbauer and infrared spectroscopy

A number of new complexes of R₂Sn^IV with adenosine, guanosine, inosine, cytidine and uridine were synthesized by reaction of ribonucleosides with diorganotin oxide in hot methanol. The complexes were characterized by infrared and ¹¹⁹Sn Mössbauer spectroscopy as O(2′), O(3′) (diorganostannylene) nucleosides; the occurence of dimers with three-co-ordinate oxygen atoms is inferred on the basis of spectroscopic data.     

Modification of ribonucleic acid by chemical carcinogens. VI. Effect of N-2-acetylaminofluorene modification of guanosine on the codon function of adjacent nucleosides in oligonucleotides

Oligonucleotides containing a guanosine residue on the 5′ or the 3′ side of tri- and tetranucleotides were prepared. The guanosine residue was modified with the chemical carcinogen N-2-acetylaminofluorene and the control and modified oligonucleotides were tested for their ability to stimulate ¹⁴C-labeled amino-acyl-tRNA binding to ribosomes. The effects of the modification are twofold. The first is that if the guanosine residue to which the drug is eovalently bound is part of a codon the oligonucleotide is completely inactive in the ribosomal binding assay. The second is that if an adenosine residue is adjacent to either the 5′ or 3′ side of the modified guanosine, as in (Ap)₃G or G(pA)₃, there is partial inhibition of ¹⁴C-labeled lysyl-tRNA binding to ribosomes. This inhibitory effect extends only to the function of the immediately adjacent adenosine since the chemical modification of guanosine residues in (Ap)₄G or G(pA)₄ did not impair their ability to code for lysine. In contrast to these findings if there is a uridine residue adjacent to the modified guanosine, as in (Up)₃G or G(pU)₃ there is no effect on ¹⁴C-labeled phenylalanyl-tRNA binding to ribosomes. Proton magnetic resonance spectra of UpG, GpU and the corresponding dinners in which the guanosine residue was modified with the drug failed to indicate a stacking interaction between the fluorene moiety and the adjacent uridine residue. This is in contrast to previous studies demonstrating a strong stacking interaction between fluorene and adjacent adenosine residues. Taken together these results indicate that acetylaminofluorene modification of guanosine next to an adenosine residue in oligonucleotide inhibits its ribosomal binding capacity. The stacking interaction with adjacent adenosine, and not with adjacent uridine residues, in oligonucleotides probably accounts for the effects observed in the ribosomal binding assay. These data are consistent with our previously described “base displacement” model.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

Partial purification and properties of a nucleoside hydrolase from Crithidia

An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl₂, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.     

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.

Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K_m value computed for inosine is 65 micromolar.

Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V_max/K_m) towards adenosine is 48 times lower than that towards inosine.

In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

     

Conformational studies on the 2′-deoxy nucleosides

Variable-temperature 220-MHz n.m.r. studies conducted on the 2′-deoxy derivatives of cytidine, thymidine, uridine, adenosine, guanosine, inosine and, to a limited extent, their 5′-phosphate disodium salts, allowed accurate proton-shift and coupling data to be obtained for the 2-deoxy-β-D-erythro-pentofuranosyl portion of the molecule. Conformational analysis, aided by the DAERM method, indicated that the sugar moiety of these molecules has a favored conformation ²V ? ²T₃ ? V₃ and an alternative favored conformation of ⁰V?⁰T₄?V₄.     

Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus

ATP and adenosine are well-known neuroactive compounds. Other nucleotides and nucleosides may also be involved in different brain functions. This paper reports on extracellular concentrations of nucleobases and nucleosides (uracil, hypoxanthine, xanthine, uridine, 2'-deoxycytidine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenosine) in response to sustained depolarisation, using in vivo brain microdialysis technique in the rat thalamus. High-potassium solution, the glutamate agonist kainate, and the Na(+)/K(+) ATPase blocker ouabain were applied in the perfusate of microdialysis probes and induced release of various purine and pyrimidine nucleosides. All three types of depolarisation increased the level of hypoxanthine, uridine, inosine, guanosine and adenosine. The levels of measured deoxynucleosides (2'-deoxycytidine, 2'-deoxyuridine and thymidine) decreased or did not change, depending on the type of depolarisation. Kainate-induced changes were TTX insensitive, and ouabain-induced changes for inosine, guanosine, 2'-deoxycytidine and 2'-deoxyuridine were TTX sensitive. In contrast, TTX application without depolarisation decreased the extracellular concentrations of hypoxanthine, uridine, inosine, guanosine and adenosine.Our data suggest that various nucleosides may be released from cells exposed to excessive activity and, thus, support several different lines of research concerning the regulatory roles of nucleosides.     

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP–Mg²⁺ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.     

Adenosine aminohydrolase inhibition in cosolvent--buffer mixtures

Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in the cooperative water structure of its environment as induced by the cosolvent dioxane. When dioxane is added to lower the dielectric constant from that of 78 of neat water to about 74, V is approximately halved, competitive inhibition by N⁶-(Δ²-isopentenyl)adenosine is virtually abolished, and competitive inhibition by the product of the reaction, i.e., inosine, is significantly decreased (K_i changes from 0.2 to 0.5 mm inosine). Yet K_m remains unaltered at 40 μm adenosine even to a dielectric constant of 66.Since both N⁶-(Δ²-isopentenyl)adenosine and inosine are competitive inhibitors, they cannot be bound by the enzyme at the same time as adenosine. The fact that substrate binding remains unaltered at dielectric constants where these inhibitors are impotent indicates that binding of these inhibitors by portions of the enzyme not directly involved in substrate binding is important. The degree of alteration of binding with increasing dioxane concentration is different for these two inhibitors, with appreciable inosine binding at mole fractions dioxane where N⁶-(Δ²-isopentenyl)-adenosine binding cannot be demonstrated. Because of this differential effect of dioxane on inosine and N⁶-(Δ²-isopentenyl)adenosine binding, it is apparent that two substances can be competitive inhibitors kinetically and yet be bound differently by an enzyme. Cosolvents may thus be useful probes for the study of enzyme inhibitor interactions. It is proposed that studies of cosolvent effects on enzyme catalysis and substrate and inhibitor binding are capable of revealing the sensitivities of these various sites to alterations in the dielectric constant of the medium and thus may be considered as models for enzyme behavior near cytoplasmic membranes in vivo.     

Reactivity of metal chelates of sulphur containing ligands towards Lewis bases. Part III. Reaction of bis(1,2-dimethyl-and 1-phenyl-1,2-ethanediylidene)bis(S-methylhydrazinecarbodithioate)NN′SS′(−2)dizinc(II) with pyridines

The reaction of the dimeric zinc(II) chelates of the type I (R₁ = R₂ = CH₃, R₁ = H, R₂ = Ph) with pyridine, 2-methylpyridine, 3-methylpyridine and 4-methylpyridine afforded the monomeric monobase adducts. The isolated adducts were characterized by their electronic and ¹H NMR spectra, and a five coordinate square pyramidal structure was tentatively assigned for these adducts.The adduct formation reaction was followed spectrophotometrically and the reaction kinetics were studied using a stopped flow technique. From the available kinetic data, as well as the measured activated parameters (ΔH^#, ΔS^#), a mechanism for the adduct formation reaction is proposed.     

Kinetics and mechanism of the oxidation of L-ascorbic acid by trisoxalatocobaltate(III) in basic aqueous solution

The kinetics and mechanism of the oxidation of L- ascorbic acid by trisoxalatocobaltate(III) were studied as a function of pH, ascorbate concentration, ionic strength and temperature in a weakly basic aqueous solution. The pH dependence of the process can be ascribed to the oxidation of the doubly deprotonated ascorbate ion for which k = 20 M⁻¹ s⁻¹ at 25 °C, ΔH^# = 34 ± 2 kJ mol⁻¹ and ΔS^# = −108 ± 7 J K⁻¹ mol⁻¹. The results are discussed in reference to literature data for this reaction in weakly acidic medium and for the oxidation by a series of other oxidants.     

Enzymatic assays of L-serine in biological material

Affinity chromatography of adenosine deaminase (EC 3.5.4.4.) on agarose-bound inosine with biospecific elution of the enzyme using linear gradients of adenosine or inosine leads via chromatographic parameters to a dissociation constant of the binary complex of K_diss = 3.5 × 10^?3m and to a binding enthalpy of ΔH = ?3.9 kcal mol^?1. These values can be explained by formation of two hydrogen bonds between immobilized inosine and the enzyme. The measurement of height equivalents of theoretical plates of the affinity column with dependence on the flow rate leads to the assumption that the velocity with which the equilibrium is reached is high compared with the flow rate; the high specificity of the affinity resin is not first of all due to a high number of theoretical plates but to the selectivity of the heterogenous enzymic reaction.