A synergistic effect of phosphate, pH and Phe159 substitution on the formycin A association to the E. coli purine nucleoside phosphorylase.

A steady-state absorption and emission spectroscopy was used to create a comprehensive work and to study the interaction of the wild type Escherichia coli purine nucleoside phosphorylase and its mutants, PNPF159Y and PNPF159A, with a potent E. coli PNP inhibitor - formycin A. The absorption and emission spectra were recorded in the presence and absence of the phosphate at the 50 mM concentration.

Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.

The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E.

Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N(4)-hydroxy-dCMP.

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.

[Role of tautomerism in the molecular mechanisms of mutagenesis].

Environment of human being usually contains a high number of environmental mutagens, which may modify chemically nucleic acid bases into promutagenic analogues. Hydroxylamine (NH2OH) is a strong mutagen which modifies cytosine and adenine to N4-hydroxycytosine and N6-hydroxyadenine, respectively. Once these analogues are present in DNA or RNA, they may cause transition point mutations by the exchange between two pairs C:G and A:T into T:A and G:C, respectively.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases.

Fluorescence anisotropy of tyrosinate anion using one-, two- and three-photon excitation: tyrosinate anion fluorescence.

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250-310 nm), two-photon (570-620 nm) and three-photon (750-930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

Background: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts.

3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization.

Analysis of proton exchange kinetics with time-dependent exchange rate.

Mass spectrometry is used to probe the kinetics of hydrogen-deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange.

Fluorescence emission properties of 8-aza analogues of caffeine, theophylline, and related N-alkyl xanthines.

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan, is highly fluorescent.

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin.

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m(7)Guo), and orthophosphate (P(i), natural cosubstrate) and its thiophosphate analogue (SP(i)), found to be a weak cosubstrate, have been studied in the pH range 5-8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK(a) 9.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves.

Interpretation of fluorescence decay kinetics in 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides: exponential dependence on the number of phosphates in the polyphosphate bridge.

The number of phosphate groups in the 5',5'-polyphosphate bridge of mRNA-cap dinucleotide analogues affects kinetics of long-range electron transfer (ET) responsible for 3-methylbenzimidazole (m(3)B) fluorescence quenching in model dinucleotides. For instance, 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides (m(3)Bp( n )G, n = 2, 3, 4) having m(3)B donor, 5'-5' polyphosphate bridge, and guanine (G) acceptor, exhibit exponential dependence of the ET rate on the number of phosphates, i.e.

A new approach to interpretation of heterogeneity of fluorescence decay: effect of induced tautomeric shift and enzyme-->ligand fluorescence resonance energy transfer.

Fluorescence decays in protein-ligand complexes are described by a new efficient model of continuous distribution of fluorescence lifetimes, and compared with multi-exponential models. Resulted analytical power-like decay function provides good fits to highly complex fluorescence kinetics. Moreover, this is a manifestation of so-called Tsallis q-exponential function, which is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence.

Photoisomerizations of N4-hydroxycytosines.

A series of N4-hydroxycytosines, unsubstituted or substituted with methyl groups at N3 or C5 atoms of the heterocyclic ring, was studied using the matrix-isolation method. Depending on the absence or presence of the methyl substituent at N3 or C5 atoms (or at both of them) the syn or anti form of the compounds (or a mixture of both forms) was trapped from the gas phase into a low-temperature matrix. Upon UV (lambda > 295 nm) irradiation of the matrixes the syn --> anti as well as the anti --> syn photoisomerization reactions were observed.

Interpretation of intramolecular stacking effect on the fluorescence intensity decay of 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides using a model of lifetime distribution.

Time-resolved fluorescence of 3-methylbenzimidazole (m(3)B) was used to study stacking interaction between base moieties in di-, tri- and tetra-phosphate analogues of 3-methylbenzimidazolyl(5'-5')guanosine (m(3)Bp( n )G, n = 2, 3, 4), using 5'-triphosphate of 3-methylbenzimidazole riboside (m(3)BTP) as reference. Fluorescence intensity decays of all compounds cannot be satisfactory fitted with single-exponential function. Although an increase of a number of exponents led to better fits, interpretation of the individual exponential terms, i.

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes.

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C.

Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene.

Purine nucleoside phosphorylases (PNPs, E. C. 2.

Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways.

The 6-oxopurine xanthine (Xan, neutral form 2,6-diketopurine) differs from the corresponding 6-oxopurines guanine (Gua) and hypoxanthine (Hyp) in that, at physiological pH, it consists of a approximately 1:1 equilibrium mixture of the neutral and monoanionic forms, the latter due to ionization of N(3)-H, in striking contrast to dissociation of the N(1)-H in both Gua and Hyp at higher pH. In xanthosine (Xao) and its nucleotides the xanthine ring is predominantly, or exclusively, a similar monoanion at physiological pH. The foregoing has, somewhat surprisingly, been widely overlooked in studies on the properties of these compounds in various enzyme systems and metabolic pathways, including, amongst others, xanthine oxidase, purine phosphoribosyltransferases, IMP dehydrogenases, purine nucleoside phosphorylases, nucleoside hydrolases, the enzymes involved in the biosynthesis of caffeine, the development of xanthine nucleotide-directed G proteins, the pharmacological properties of alkylxanthines.

Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence.

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P(i), substrate). Formation of enzyme-FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P(i).

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases.

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP).

Interpretation of fluorescence decays using a power-like model.

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase.

Structural properties of the neutral and monoanionic forms of xanthosine, highly relevant to their substrate properties with various enzyme systems.

The monoanions of the 6-oxopurines guanine (Gua) and hypoxanthine (Hx), and their nucleosides, pKa approximately 9 due to dissociation of the N(1)-H, are predominantly in their neutral forms at physiological pH. By contrast, the monoanions of the 6-oxopurine xanthine (Xan) and xanthosine (Xao), were long ago proposed to involve dissociation of the N(3)-H, with pKa values of 7.5 and 5.

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human, and the Drosophila melanogaster, deoxyribonucleoside kinases.

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP).

Inorganic tripolyphosphate (PPP(i)) as a phosphate donor for human deoxyribonucleoside kinases.

Inorganic tripolyphosphate (PPP(i)) and pyrophosphate (PP(i)) were examined as potential phosphate donors for human deoxynucleoside kinase (dCK), deoxyguanosine kinase (dGK), cytosolic thymidine kinase (TK1), mitochondrial TK2, and the deoxynucleoside kinase (dNK) from Drosophila melanogaster. PPP(i) proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not dAdo, as acceptor substrate, illustrating also the dependence of donor properties on acceptor. Products of phosphorylation were shown to be 5(')-phosphates.