Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

Phthalazinone inhibitors of inosine-5′-monophosphate dehydrogenase from Cryptosporidium parvum

Cryptosporidium parvum (Cp) is a potential biowarfare agent and major cause of diarrhea and malnutrition. This protozoan parasite relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for the production of guanine nucleotides. A CpIMPDH-selective N-aryl-3,4-dihydro-3-methyl-4-oxo-1-phthalazineacetamide inhibitor was previously identified in a high throughput screening campaign. Herein we report a structure–activity relationship study for the phthalazinone-based series that resulted in the discovery of benzofuranamide analogs that exhibit low nanomolar inhibition of CpIMPDH. In addition, the antiparasitic activity of select analogs in a Toxoplasma gondii model of C. parvum infection is also presented.     

Synthesis of the inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitors

Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) is important molecular target for potential anticancer, antiviral, antibacterial and immunosuppressive agents. A lot of compounds were obtained to establish their activity toward this enzyme, and to improve therapeutic properties of IMPDH inhibitors used as the drugs. Some of the recently reported analogs exhibited promising results during in vitro and in vivo examinations in comparison to substances applied in clinic. In this review, we describe synthesis and biological activity evaluations of the newly designed IMPDH inhibitors.     

Radiation chemical studies of sensitization by 5-bromouridine-5′-monophosphate (5-BrUMP)

Summary This study was undertaken to investigate the mechanism of chemical radiosensitization by halogenated bases incorporated into DNA. Radiation-induced base and sugar-phosphate backbone damage to 5-bromouridine-5-monophosphate (5-BrUMP) was monitored using a flow system connected in series with a recording spectrophotometer, a bromide (Br^–)-specific ion analyzer and a Technicon auto-sampler. The system was used to assay loss of UV-absorbing 5,6 double-bond, release of Br^– and inorganic phosphate (Pi) release using an automated colorimetric method, as a function of gamma-ray dose. Results obtained with radical scavengers indicate that, unlike non-halogenated nucleotides where the hydroxyl radical (· OH) is the principal damaging species, 5-BrUMP is damaged by the hydrated electron (e _aq ^– ), hydrogen atom (H·) and · OH, producing a high yield of base damage and Br^– and Pi release in anoxia. Another novel feature of 5-BrUMP radiolysis is that oxygen, by convertinge _aq ^– and H· to the unreactive superoxide radical anion (0 ₂ ^– ), has a protective effect on both base and phosphate ester damage. Under · OH-scavenging conditions, where the radiation yield of reductive debromination is 3.8, there is some Pi release, suggesting the possibility of intramolecular hydrogen atom transfer from the sugar ring to the 5-uracilyl radical and subsequent sugar-phosphate bond cleavage. This hypothesis is supported by the action of oxygen and thiols in modifying thee _aq ^– -mediated sugar-phosphate damage.     

Cyclic adenosine 3′,5′-monophosphate and germination of sporangiospores from the fungus Mucor

Cyclic adenosine 3,5-monophosphate (cAMP) metabolism was examined in germinating sporangiospores of Mucor genevensis and Mucor mucedo. Exogenous cAMP prevented normal hyphal development from sporangiospores. Internal pools of cAMP fluctuated profoundly during development. Spherical growth of the spores was characterized by large pools of cAMP whereas germ tube emergence and hyphal elongation were characterized by small pools of cAMP. These observations suggest a possible role for cAMP in sporangiospore germination. Adenylate cyclase activities fluctuated significantly during germination with maximum values attained during spherical growth. In contrast, cAMP phosphodiesterase activities remained constant throughout germination. Internal cAMP levels may therefore be regulated by adjustment of adenylate cyclase activities. The binding of cAMP by soluble cell proteins was measured. cAMP-binding activity changed greatly during germination. Dormant and spherically growing spores possessed the highest activities. Developing hyphae contained the lowest activities. Use of the photoaffinity label, 8-azido-[^32P]cAMP, in conjunction with sodium dodecyl sulfate-polyacrylamide-gel electrophoresis allowed the identification of a small population of morphogenetic-stage-specific proteins which bind cAMP and may be of regulatory significance to development.     

Sustainable synthesis of uridine-5′-monophosphate analogues by immobilized uracil phosphoribosyltransferase from Thermus thermophilus

Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70–100 °C) and in the pH interval 6–8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/g_support, retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5′-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5′-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.     

Properties of 5′-nucleotidase from nodules of pigeonpea (Cajanus cajan)

5′-Nucleotidase from pigeonpea nodules has been resolved into two forms, N-I and N-II, having M,s of 52 000 and 119 000, respectively. Both forms had pH optima in the acidic range (between pH 5.2 and 5.7) with either CMP, GMP, XMP, IMP or AMP as the substrate. Up to pH 6.6, both forms showed higher activity with CMP followed by GMP, XMP, IMP and AMP, respectively. However, the activity changed with pH in the alkaline range making the enzyme relatively more active with purine nucleotides. Neither of the forms had a requirement for any of the metal ions tested. Fe³⁺ inhibited the enzyme activity; the inhibition at 5, 10 and 15 mM concentrations being 11, 43 and 47%, respectively with N-I and 14,47 and 52%, respectively with N-II. K_m values for AMP, IMP, GMP, CMP and XMP were 0.10, 0.18, 0.40, 0.40 and 0.77 mM, respectively with N-I and 0.12, 0.20, 0.40, 0.40 and 0.99 mM, respectively with N-II. The enzyme was inhibited non-competitively by adenosine and inosine; K_i values being 1.78, 0.25 and 0.30; 3.50, 2.12 and 0.75 mM, respectively with AMP, IMP and XMP as the substrate.     

Crystal structure of a new form of copper(II)—inosine 5′-monophosphate—2,2′-dipyridylamine ternary complex

Two crystalline forms of the [Cu(II) (IMP) (DPA) (H₂O)]₂·nH₂O (IMP=inosine 5′-monophosphate, DPA=2,2′-dipyridylamine) complex were obtained from aqueous solution at pH=6.2. The crystals of the two forms belong to the monoclinic system, space group P2₁. The cell parameters are: a=9.445(2), b=33.902(4), c=7.802(2) Å, β=90.48(2)°, Z= 2, D_c=1.69g cm⁻³ and μ(Mo Kα) = 10.49cm⁻¹ (form α, n=4), and a=7.828(2), b=18.552(3), c=17.378(3) Å, β=91.16(2)°, Z=2, D_c=1.66 g cm⁻³, μ(Mo Kα) = 10.40 cm⁻¹ (form β, n=3.62). Bau and coworkers reported the preparation of form α by vapor diffusion of CH₃CN into aqueous solution containing Cu(NO₃)₂, Na₂IMP and DPA in a 1:1:1 molar ratio and the analysis of the compound by single crystal X-ray diffraction [1].Intensities for 3412 reflections were collected from a crystal of form β in the present work. Graphite-monochromatized Mo Kα radiation was employed. The structure was refined to final R and R_w values of 0.1000 and 0.1115 respectively. The dimeric units contain two copper ions in square-pyramidal coordination polyhedra. Each polyhedron consists of two nitrogen atoms of DPA, two oxygen atoms from two phosphate groups and a water molecule in the axial position. A statistical disorder was found in a nucleotide moiety of the dimer. Two sets of atomic positions corresponding to the purine system were refined with site occupation factors of 0.62(1) and 0.38(1) respectively. Also the ribose ring shows a disorder with two possible conformations. The puckering mode of the prevailing conformation is C(3′)-endo. In the other nucleotide molecule of the dimer the furanose puckering mode is C(3′)-endo. The rotation around the glycosyl linkages can be described as ‘anti’ in the structure of form β. The C(4)N(9)C(1′)O(4′) torsion angle values are −97(2) and −94(3)° for the disordered nucleotide molecule and +91(2)^o for the other nucleotide moiety. Strong intermolecular DPADPA and purine-purine stacking interactions stabilize the crystal lattice. The differences on the nucleotide conformation between the structure of form α and form β can probably be ascribed to differences in the hydrogen bonds and stacking interactions.     

Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-beta-d-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP>AMP. NAD(+) did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in lambda(max.) of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A(290) with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2-3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[(14)C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[(14)C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.     

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

• 1.1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
• 2.2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
• 3.3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

     

Monoamine regulation of adenosine 3′,5′-monophosphate in homogeneous neuronal cultures from chick brain hemispheres

The effect of isoproterenol, norepinephrine, dopamine, and serotonin on the accumulation of adenosine 3,5-monophosphate (cAMP) was studied in homogeneous neuronal cultures from 8-day chick embryo hemispheres. Among the catecholamines, isoproterenol had a more pronounced effect on the accumulation of cAMP. Norepinephrine and dopamine were considerably less potent and serotonin was ineffective. The response of neuronal cells to isoproterenol was inhibited by propranolol, suggesting that the cAMP increase was mediated by -adrenergic receptors. Maximally effective concentration of isoproterenol (10 M) produced a 2.5-fold increase in cAMP content which is in contrast to the much greater cAMP response elicited by isoproterenol in chick brain tissue. These results suggest that in chick embryo hemispheres the nonneuronal cells are the major sites of the effect of -adrenergic agonists. The low responsiveness of the cAMP-generating system found in neuronal cultures in interpreted as reflecting either the number of -adrenergic receptors or the regulation of -adrenergic receptors and adenylate cyclase at the membrane level.     

The incorporation of cytidine 5′-monophosphate and other ribonucleotides by an enzyme preparation from rat-liver microsomes

1. An enzyme preparation from rat-liver microsomes incorporated all four ribonucleotides from the corresponding triphosphates into ribosomal RNA. The reaction was Mn(2+)-dependent, but UMP incorporation also occurred in the presence of Mg(2+). 2. The incorporation of any one ribonucleotide was inhibited by the presence of the other three ribonucleoside triphosphates and by denatured DNA. 3. The product of the reaction consisted of short chains of homopolymer attached to the primer ribosomal RNA. 4. ;Soluble' RNA, synthetic polyribonucleotides, and oligoribonucleotides were also effective primers for CMP incorporation. 5. When phosphodiesterase-treated ;soluble' RNA was the primer, CMP was incorporated into positions usually occupied by the normal terminal trinucleotide sequence of intact ;soluble' RNA, but the enzyme did not synthesize a specific terminal sequence consisting of a defined number of CMP residues.     

Stable operation of microbial fuel cells at low temperatures (5–10 °C) with light exposure and its anodic microbial analysis

Performances of microbial fuel cells (MFCs) were studied at 5–10 and 25–30 °C. Results showed stable operation of the MFCs at low temperatures with only slight reductions of voltage and power generation (11 versus 14 % for double-chamber MFC, while 14 versus 21 % for single-chamber MFC, 1,000 Ω) compared to those at mesophilic temperatures. MFCs operated at low temperatures showed lower COD removal rates accompanied by higher coulombic efficiencies (CEs). PCR-DGGE analysis revealed that psychrotrophic microbes (mainly Arcobacter, Pseudomonas, and Geobacter) dominated on anodes of the MFCs at low temperatures. Interestingly, light-induced red substances appeared on anode of the MFCs operated at low temperature and were proven to be the main anodic microbes (Arcobacter and Pseudomonas). Co-existence of the aforementioned microbes could assist stable low-temperature operation of the MFCs. Cyclic voltammetry analysis supported the results of the CE and DGGE. Stable performance of MFCs at low temperatures might be achieved by the control of anodic bacteria.     

Gas chromatographic-mass spectroscopic identification and quantification of cyclic guanosine-3′: 5′-monophosphate in maize seedlings (Zea mays)

Gas chromatographic-mass spectroscopic evidence is presented for the presence of guanosine-3′: 5′-monophosphate (cGMP) in maize seedlings. The amount of cGMP (35–72 pmol g^-1 fresh weight) was quantified as a tetra-silyl derivative using gas-chromatographic detection with reference to a silylated standard of authentic cGMP. Gas-chromatographic separation of tri-silyl adenosine-3′: 5′-monophosphate and tetra-silyl cGMP is demonstrated.     

Cobalt-requiring 5′-deoxy-5′-methylthioadenosine nucleosidase from soybean (Glycine max) cotyledon

5??-Deoxy-5??-methylthioadenosine nucleosidase (MTA nucleosidase, EC 3.2.2.9) was purified from soybean (Glycine max) cotyledon. The nucleosidase was a trimer consisting of three identical subunits with a molecular mass of 59.5?kDa. The nucleosidase was a cobalt-requiring enzyme for its catalytic function. The enzymatic activity increased in a dose-dependent manner in the presence of cobalt. Cobalt was bound to the nucleosidase with a stoichiometry of 1 equivalent of cobalt/subunit. A thiol group-specific reagent reduced the enzymatic activity. Four cysteinyl residues of each subunit are considered to play an important role in binding cobalt.     

Cis-diammineplatinum(II) forms a macrochelate with 2′-deoxycytidine 5′-monophosphate (dCMP2–)! Reactivity and acid-base properties of cis-Pt(NH3)2(dCMP)

 The synthesis of cis-Pt(NH₃)₂(dCMP) is reported and by various physico-chemical methods it is demonstrated that it is a macrochelate in which Pt(II) is bound simultaneously to the N3 site of cytosine in dCMP^2– and to a phosphate-oxygen atom. According to the NOESY spectra (cross-peaks between cytosine H6 and H2′ and H3′) the cytosine ring adopts an anti orientation. Highly unusual is the significant (1 ppm) downfield shift of the sugar proton H5″ in the ¹H-NMR spectrum and the sensitivity of the cytosine H6 resonance on the protonation state of the phosphate group. Based on these three features a geometry for the macrochelate is proposed. The compound is a major product of the reaction of cis-[Pt(NH₃)₂(H₂O)₂]²⁺ with dCMP^2– at neutral pH, but it even forms at pH 5. By applying pD-dependent NMR spectroscopy (¹H, ³¹P) and potentiometric pH titration, it is demonstrated that the Pt-coordinated phosphate group can be protonated (pK _a/1=3.21±0.10 and 3.31±0.05, respectively), and ¹H- and ³¹P-NMR spectra also indicate deprotonation (pK _a/2=13.35±0.25) of the exocyclic amino group of the cytosine moiety. The metal ion binding affinity of cis-Pt(NH₃)₂(dCMP) is very small, as shown for Cu²⁺ (log K<0.6). The cis-Pt(NH₃)₂(dCMP) complex reacts with nucleosides and nucleotides (L′) by losing its chelate structure and forming mixed ligand complexes, cis-Pt(NH₃)₂(dCMP)(L′); this means that the phosphate group is released from the coordination sphere of Pt(II), indicating that the Pt(II)-O(phosphate) bond is not very strong. Received: 23 October 1997 / Accepted: 17 February 1998