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Novel multifunctional biomimetic injectable hybrid systems were synthesized. The physicochemical as well as biological in vitro and in vivo tests demonstrated that they are promising candidates for bone tissue regeneration. The hybrids are composed of a biopolymeric collagen/chitosan/hyaluronic acid matrix and amine group-functionalized silica particles decorated with apatite to which the alendronate molecules were coordinated. The components of these systems were integrated and stabilized by cross-linking with genipin, a compound of natural origin. They can be precisely injected into the diseased tissue in the form of a viscous sol or a partially cross-linked hydrogel, where they can serve as scaffolds for locally controlled bone tissue regeneration/remodeling by supporting the osteoblast formation/proliferation and maintaining the optimal osteoclast level. These materials lack systemic toxicity. They can be particularly useful for the repair of small osteoporotic bone defects.

Replacing water with dimethyl sulfoxide (DMSO) completely reshapes the free-energy landscapes of solvated proteins. In DMSO, a powerful hydrogen-bond (HB) acceptor, formation of HBs between backbone NH groups and solvent is favored over HBs involving protein’s carbonyl groups. This entails a profound structural disruption of globular proteins and proteinaceous aggregates (e.g., amyloid fibrils) upon transfer to DMSO. Here, we investigate an unusual DMSO-induced conformational transition of β2-amyloid fibrils from poly-l-glutamic acid (PLGA). The infrared spectra of β2-PLGA dissolved in DMSO lack the typical features associated with disordered conformation that are observed when amyloid fibrils from other proteins are dispersed in DMSO. Instead, the frequency and unusual narrowness of the amide I band imply the presence of highly ordered helical structures, which is supported by complementary methods, including vibrational circular dichroism and Raman optical activity. We argue that the conformation most consistent with the spectroscopic data is that of a PLGA chain essentially lacking nonhelical segments such as bends that would provide DMSO acceptors with direct access to the backbone. A structural study of DMSO-dissolved β2-PLGA by synchrotron small-angle X-ray scattering reveals the presence of long uninterrupted helices lending direct support to this hypothesis. Our study highlights the dramatic effects that solvation may have on conformational transitions of large polypeptide assemblies.

Background: Purine-nucleoside phosphorylase (PNP) is known as a tool for the synthesis of various nucleosides and nucleoside analogues. Mechanism, properties, molecular diversity and inhibitors of PNP, particularly these of pharmacological significance, are briefly characterized. Methods: UV and fluorescence spectroscopy was used for kinetic experiments, and HPLC chromatography for product analyses. Results: Applications of various forms of PNP to synthesis of selected fluorescent nucleosides, particularly ribosides of 1,N6-ethenoadenine and various 8-azapurines (triazolo[4,5-d]pyrimidines) are reviewed. Different specificity of various PNP forms is described towards nucleobase and analog substrates as well as variable ribosylation sites observed in some reactions, with a possibility to further modify these features via the site-directed mutagenesis. Conclusion: Present and future applications of the fluorescent or fluorogenic ribosides are discussed, with particular emphasis on biochemical and clinical analyses with improved sensitivity.

HSP90 is constantly externalized on the surface of human macrophages; selective inhibition of this HSP90 pool suppresses the macrophages' response to PAMPs. Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on M?. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived M?s (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (a and ß) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus(SA) and Porphyromonas gingivalis(PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a “signaling complex chaperone,” with activity that is essential for cytokine response but not for target engulfment by M?.

ABSTRACTGingipains are cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. They consist of arginine-specific (HRgpA and RgpB) and lysine-specific (Kgp) proteinases. Gingipains strongly affect the host defense system by degrading some cytokines, components of the complement system, and several immune cell receptors. In an in vitro model, gingipains were shown to degrade soluble tumor necrosis factor alpha (TNF-α). However, since membrane TNF-α shows strong biological activity, especially in local inflammatory lesions, it was worth investigating whether gingipains might also destroy membrane TNF-α and limit its biological activities. To avoid a possible influence of gingipains on ADAM17, the secretase of TNF-α, the majority of experiments were performed using ADAM17−/−fibroblasts stably transfected with cDNA of human pro-TNF-α (ADAM17−/−TNF+). Arginine-specific gingipains (Rgp's) strongly diminished the level of TNF-α on the cell surface as measured by flow cytometry, and this process was not accompanied by an increased concentration of soluble TNF-α in the culture medium. Degradation of membrane TNF-α by Rgp's correlated with a strong decrease in TNF-α-mediated biological activities of ADAM17−/−TNF+cells. First, the activation state of transcription factor NF-κB was suppressed; second, the cells were no longer able to induce apoptosis in HL-60 cells. Kgp was also able to cleave membrane TNF-α, but its effect was much weaker than that of Rgp's. Gingipains also limited the binding of native TNF-α to the target cells. Thus, gingipains are able not only to cleave soluble TNF-α but also to destroy the membrane form of the cytokine, which may additionally dysregulate the cytokine network.

FTY720 induces rapid cell surface expression of HSP27, which coincides with atypical cell death sensitive to inhibitors of apoptosis, necroptosis, or NADPH oxidase. The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod‐induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain‐like protein. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase‐8. Fingolimod‐induced neutrophil death is independent of sphingosine‐1‐phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase‐8 [Z‐Ile‐Glu(O‐Me)‐Thr‐Asp(O‐Me)‐fluoromethyl ketone], receptor‐interacting protein kinase 1 (necrostatin‐1), receptor‐interacting protein kinase 3 (necrosulfonamide), and heat shock protein 90 [geldanamycin and 17‐(dimethylaminoethylamino)‐17‐demethoxygeldanamycin]. Furthermore, NADPH oxidase 1 inhibition with diphenyleneiodonium chloride protects neutrophils against fingolimod‐mediated cell death. Overall, these observations suggest that fingolimod acts through a mechanism involving the necrosome signaling complex and the oxidative stress machinery.

The enhanced green fluorescent protein (EGFP) is broadly used as an intracellular fluorescent marker. Its numerous applications require a comprehensive understanding of EGFP folding properties, as well as the properties of EGFP-derived mutants.Herein, we report on the spectroscopic characterization of two single-cysteine mutants, C48S-EGFP and C70S-EGFP, which were prepared to monitor the protein folding dynamics by EPR, specifically in the close vicinity of both cysteine residues.Chromophore absorption, excitation and emission spectra for these mutants were similar to those for EGFP. Both single-cysteine mutants were more susceptible to aggregation than EGFP in E. coli. C48S-EGFP exhibited similar resistance to chemical denaturation as EGFP. In contrast, C70S-EGFP was less resistant to denaturation in guanidine hydrochloride and its folding was less efficient. Hence, C48S-EGFP seems to be a more suitable probe for EPR measurements than C70S-EGFP.

AbstractCirculating neutrophils and monocytes form the first line of cellular defense against invading bacteria. Here, we describe a novel and specific mechanism of disabling and eliminating phagocytes by Staphylococcus aureus. Staphopain B (SspB) selectively cleaved CD11b on phagocytes, which rapidly acquired features of cell death. SspB-treated phagocytes expressed phosphatidylserine as well as annexin I and became permeable to propidium iodide, thus demonstrating distinctive features of both apoptosis and necrosis, respectively. The cell death observed was caspase and Syk tyrosine kinase independent, whilst cytochalasin D efficiently inhibited the staphopain-induced neutrophil killing. Neutrophil and monocyte cell death was not affected by integrin clustering ligands (ICAM-1 or fibrin) and was prevented, and even reversed, by IgG. This protective effect was dependent on the Fc fragment, collectively suggesting cooperation of the CD16 receptor and integrin Mac-1 (CD11b/CD18). We conclude that SspB, particularly in the presence of staphylococcal protein A, may reduce the number of functional phagocytes at infection sites, thus facilitating colonization and dissemination of S. aureus.Copyright © 2008 S. Karger AG, Basel

Fluorapophyllite, (K0.96Na0.03)0.99Ca3.96 (Si7.95Al0.04P0.03)8.02O19.98(F0.83OH0.17) · 8.02 H2O has been found in the Miedzyrzecze sill. The apophyllite crystals were collected in the complex veins cross cutting small pectolite skarn body at the top of picrite sill. Apophyllite from Miedzyrzecze exhibits no signs of abnormal optical properties; it is tetragonal with lattice parameters: a = 8.974 (2) Å, c = 15.798 (6) Å. The dehydration behaviour of apophyllite from Mieedzyrzecze was studied by means of X-ray diffraction, thermal analysis and infrared spectroscopy; also its dielectric properties were measured. The DTA curve shows two distinct endothermic peaks connected with irreversibly removal of water molecules from K+ (OH2)8 polyhedron, centred at about 348 and 454°C, respectively. All analytical data obtained confirm suggestion, that amorphous layers on the outer parts of apophyllite grains form a diffusion barrier causing moderation of H2O molecule migration and the second endothermic peak origin. The ring diffusion of H2O starts at about 160 °C, when the water molecules begin to migrate from the outside parts of apophyllite grains through 4- and 8- membered rings along [001] direction. Expulsion of the water molecule from the K, Ca sheets causes initiation of the non-diffracting amorphous phase origin. The subsequent water loss gives occasion to permanent distortion of sillicate layers with simultaneous K and Ca coordination collapse. The IR data obtained was useful to discuss the structural property of amorphous form of dehydrated apophyllite.

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10) is up-regulated, while production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti-IL-10, suggested that annexin V-negative monocytes are the predominant source of IL-10. Analysis of sorted populations of monocytes indicated that the increase in IL-10 synthesis appears to result from direct interactions between non-apoptotic and apoptotic cells at the time of stimulation. Also non-apoptotic, freshly isolated monocytes produced more IL-10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte-derived macrophages did not produce more IL-10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V-positive monocytes CD4-positive memory T cells produce less IFN-γ upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti-IL-10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 μg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 μg/ml-0.29 mg/ml). At concentrations below 0.9 μg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (mGuo) with a catalytic rate constant 1.3×10 s as compared with the rate of 38 s for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of mGuo, 70 s, is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 μM-30 mM phosphate is consistent with only one dissociation constant for phosphate, K d =220±120 μM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 μM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for "low molecular mass" PNPs, except for its unusual stability at pH 11. Purine bases, α- d -ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N -methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 μM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K d =0.46 μM for Gua, 3.0 μM for Hx, and 60 μM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K d =270 μM, but existence of a very weak binding site with K d >50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker ( K d =1.7 μM) than to the free enzyme ( K d =0.46 μM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis. The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.

Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions. Previously, we described that human peripheral blood MO consist of a major subset of Fcγ-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity. Recently, we could show that each subset itself further differs in the expression of the Fcγ-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16+subsets (CD64+or CD64-) with high T cell stimulation capacity and two CD16-subsets (CD64+or CD64-) with low accessory activities. In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria (S. aureusandE. coli) and in the generation of reactive oxygen species. Both CD64+subsets (CD16+or CD16-) exhibit high phagocytic activity accompanied by intracellular superoxide induction. Luminal-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations. Phagocytic activity and modest CL response was shown in CD64-/CD16+but not in CD64-/CD16-cells, indicating that each subset except for CD64-/CD16-cells may engulf bacteria and exhibit MPO activity. Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens.

Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5 degrees C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.

ABSTRACTHuman peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect onS. aureus-induced apoptosis. Exposure of monocytes to 41.5°C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureusimmediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.

The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. has been determined by X-ray crystallography. The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a = 64.1 A˚, b = 108.9 A˚, c = 119.3 A˚ and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 °C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A˚ resolution. The ternary “dead-end” complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine. Data were collected at 100 K with CuKα radiation, and the three-dimensional structure refined at 2.4 A˚ resolution.Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central β-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail.In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed. This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs.The phosphate binding site is located between two conserved β- and γ-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1· · ·Glu204 Oε1· · ·guanine NH2· · ·Glu204 Oε2. The exocyclic O6 of the base is bridged via a water molecule to Asn246 Nδ, which accounts for the inhibitory, but lack of substrate, activity of adenosine.An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis. This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP.

Substrate/inhibitor properties of purine nucleoside phosphorylase (PNP), isolated from human lung and kidney tumour tissues, have been characterised and compared with those of the enzyme from the corresponding normal organs.

Without Abstract:

Trimeric calf spleen purine nucleoside phosphorylase has been complexed with hypoxanthine via phosphorolysis of inosine in the presence of phosphate. The resulting “Michaelis” complex (three hypoxanthine molecules per trimer), presumed to be formed under these conditions, crystallized in the cubic space group P213, with unit cell dimension a = 94.11 A˚ and one monomer in the asymmetric crystal unit; the biologically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structure has been determined by molecular replacement and refined at 2.15 A˚ resolution to an R-value of 0.18.In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of Glu201 and Asn243, as well as one integral water molecule located in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base positions N-3 and N-9 with an adjacent pocket, which presumably is the phosphate-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxanthine are saturated by hydrogen- bonding to protein side-chains or integral water molecules.Purine nucleoside phosphorylase isolated form human tissues is a primary target for chemotherapeutic intervention, and the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolution structure presented here may serve for design of inhibitors with potential pharmacological applications.

Quantitative structure-activity relationships (QSAR) for a series of N(7)-substituted guanosines as substrates for calf spleen purine nucleoside phosphorylase (PNP) were developed, and compared with those for acid hydrolysis of these analogues. There is no correlation between the rates for enzymatic phosphorolysis and acid hydrolysis, indicating that for the enzymatic reaction labilization of the glycosidic bond is not the only, nor the predominant, effect of N(7)-substitution. Multiple regression analysis of the enzymatic process revealed that optimal substrate properties (minimal Michaelis constant) are associated with the Taft electronic constant equal zero and a substituent size, parametrized by the Taft steric constant, smaller than that for a methyl group. These results support the hypothesis of protonation of the N(7)-position of the base by the enzyme as a catalytic mechanism for calf spleen PNP.

A new continuous fluorimetric assay for AMP deaminase activity is described. The m ethod makes use of a fluorescent analog of 5'-AMP, formycin-5'-phosphate (5'-FM P), which undergoes deam ination to formycin B-5'-phosphate, not fluorescent at neutral pH. The pH -dependence for deamination of 5'-FMP is similar to that for 5'-AMP, but shifted about 0.2 units to more acidic pH. Deamination of 5'-FM P may also be followed spectrophotometrically at 306 nm, permitting better assays of crude extracts. Some kinetic results obtained by means of the new method for AMP deaminase from chick and rabbit skeletal muscle are presented. In particular it was found that the natural product of deamination, 5'-IMP exhibited allosteric inhibition of the chick enzyme with K, values 1.6mM , 1.2mM and 1.0mM at pH 5.8, 6.5 and 7.3, respectively. Activation by diadenosine tetraphosphate, Ap4A , reported for mouse muscle AMP deaminase, has not been noted for the chick enzyme. Inhibition by the transition state analogs, coformycin and 2'-deoxycoformycin, was observed for both rabbit and chick deaminases with Kivalues ~ 1 µM and ~ 1.6 µM respectively. Kinetic data for coformycin-5'-phosphate show it to be a tight-binding inhibitor with K, < 0.6 × 10-9M as compared to 1 × 10-9m for 2'-deoxycoformycin-5'-phosphate.

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzym es. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonu cleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to proton ation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a don or and O6as an acceptor. Alternatively N(1)-H and C(2)-NH2, may serve as donors for hydrogen bonds with a glutam ate residue.

The previous finding that 7-methylinosine (m7Ino) and 7-methylguanosine (m7Guo) are excellent, as well as fluorescent, substrates for calf spleen purine nucleoside phosphorylase has been extended to include a series of 7-alkylguanosines with higher alkyl groups (ethyl, propyl, butyl, isopropyl, isobutyl, benzyl). All of these are good substrates, with increases in Km compensated for by corresponding increases in Vmax, and excluding the ring N-7 as a binding site. Both m1Ino and m1Guo are neither substrates nor inhibitors of this enzyme, implicating the ring N-1 as a binding site. Included also are guanosine analogues with C-8 substituents (alpha-hydroxyisopropyl, methyl, bromo, chloro, amino) with known populations of syn and anti conformers about the glycosidic bond; while these did not unequivocally point to involvement of only one form, efficiency of phosphorolysis appeared to be correlated with a conformation in the anti region. The influence of various substituents is also considered in relation to steric and electronic effects. Kinetic parameters for all the foregoing have been determined fluorimetrically and/or spectrophotometrically from initial velocities and/or continuous monitoring of phosphorolysis including Ki values for inhibition by the base moieties. Furthermore, kinetic data demonstrated that, in striking contrast to the mammalian enzyme, both m1Ino and m1Guo are almost as good substrates as the parent nucleosides for a bacterial (Escherichia coli) enzyme, suggesting that the ring N-1 is not a binding site for the latter.

The ternary complex of purine nucleoside phosphorylase from E. coli with formycin B and a sulphate or phosphate ion crystallized in the hexagonal space group P6122 with unit cell dimensions a = 123.11, c = 241.22 A˚ and three monomers per asymmetric unit. The biologically active hexamer is formed through 2-fold crystallographic symmetry, constituting a trimer of dimers. High-resolution X-ray diffraction data were collected using synchrotron radiation (Daresbury, England). The crystal structure was determined by molecular replacement and refined at 2.1 A˚ resolution to an R-value of 0.196.There is one active centre per monomer, composed of residues belonging to two subunits of one dimer. The phosphate binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighbouring subunit), Ser90 and Gly20. It is occupied by a sulphate or phosphate anion, each oxygen atom of which accepts at least two hydrogen bonds or salt-bridges. The sulphate or phosphate anion is also in direct contact with the ribose moiety of formycin B. The ribose binding site is composed of Ser90, Met180, Glu181 and His4, the latter belonging to the neighbouring subunit. The base binding site is exposed to solvent, and the base is unspecifically bound through a chain of water molecules and aromatic-aromatic interactions. In all monomers the nucleosides are in the high syn conformation about the glycosidic bonds with χ in the range 100 to 130°. The architecture of the active centre is in line with the known broad specificity and the kinetic properties of E. coli PNP.

Calf spleen purine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)‐acycloguanosine inhibitor crystallized in the cubic space group P213, with unit cell dimension and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three‐fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 Å to an R‐factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of nucleosides.

Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface.

The commercially available enzyme purine nucleoside phosphorylase (PNP) from Cellulomonas sp. was purified by ion‐­exchange chromatography, partially sequenced and crystallized in two different crystal forms using the hanging‐drop vapour‐diffusion technique. Crystal form Agrows as polyeders and/or cubes in the cubic space group P4232 with unit‐cell dimension a= 162.5 Å. Crystal form Bappears as thick plates in the space group P212121with unit‐cell dimensions a= 63.2, b= 108.3 and c= 117.4 Å. Both crystal forms contain three monomers (one trimer) in the asymmetric unit.

9-(5′,5′-Difluoro-5′-phosphonopentyl)-9-deazaguanine (DFPP-DG) and its related analogues were designed as multi-substrate analogue inhibitors against purine nucleoside phosphorylase (PNP) on the basis of the X-ray crystallographic data obtained for the binary complex of 9-(5′,5′-difluoro-5′-phosphonopentyl)guanine (DFPP-G) with calf-spleen PNP. One of these analogues, homo-DFPP-DG was found to be a very potent PNP inhibitor at an intracellular Pi concentration (about 1 mM phosphate).

AbstractA series of 2-chloro-6-aryloxy- and 2-chloro-6-alkoxyarylpurines was synthesized and their kinetic properties in the purine nucleoside phosphorylase (PNP) system were determined. All compounds showed inhibitory activity (IC50in the range 0.5-76 μM) vs.hexameric (“high-molecular weight”) PNP from E. coli.By contrast, no inhibition vs.trimeric CellulomonasPNP was detected.

AbstractDissociation constants and stoichiometry of binding for interaction of Cellulomonas sp.purine nucleoside phosphorylase with its substrates: inosine/guanosine, orthophosphate, guanine/hypoxanthine and D-ribose-1-phosphate were studied by kinetic and spectrofluorimetric methods.

AbstractAssociation between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.

Kinetic parametrs for enzymatic cleavage of the glycosidic bond (phosphorolysis) of ten N(7)-substituted guanosines were determined, and used to establish a structure-activity relation for the Michaelis constants. Results were compared with those for acid-catalyzed cleavage of the glycosidic bonds, and are consistent with a mechanism for phosphorolysis via protonation of the purine ring N(7).

GFP (Green Fluorescent Protein) is well known for its unique chromophore which is formed by autocatalytic cyclization of a polypeptide backbone of Ser65, Tyr66 and Gly67, and is able to emit green visible light. Due to unusual chromophore responsible for the fluorescence GFP and its mutants (e.g., EGFP) have become widely used reporter proteins in molecular biology and biotechnology. GFP can easily be fused to any protein of interest and co-expressed in cells; the GFP fluorescence is then used to visualize the distribution, transport and aggregation of the protein in the cell. However, GFP has a tendency to aggregate itself, and also formation of its chromophore critically depends on the presence of reducing agents. Therefore we have undertaken spectroscopic kinetic studies of EGFP folding and aggregation as a function of pH, and in the presence of various reducing agents, to study the competition between these two processes. The best conditions for folding of EGFP provides BME as a reducing agent. Aggregation of EGFP depends strongly on pH, and on the concentration of the protein. The careful control experiments must therefore be performed during investigations of proteins fused with EGFP, especially at pH lower than 7.

The steady-state fluorimetric titration curves for trimeric purine nucleoside phosphorylase (PNP) by two ligands, were analysed using the DynaFit program. Results of this analysis indicate that three binding sites of PNP molecule interact with each other and that the character of this interaction is different for both ligands. The DynaFit program is very useful in studies of oligomeric proteins, but for detection of non-interacting sites some independent tests are necessary.

9-(5′,5′-Difluoro-5′-phosphonopentyl)-9-deazaguanine (DFPP-DG) and its related analogues were designed as multi-substrate analogue inhibitors of purine nucleoside phosphorylase (PNP) on the basis of the X-ray crystallographic data obtained for the binary complex of 9- (5′,5′-difluoro-5′-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. One of these analogues, homo- DFPP-DG was found to be a very potent PNP inhibitor at an intracellular (∼ 1 mM) phosphate concentration.

A simple and in principle universal method is proposed for measuring enzyme/ligand dissociation constants. The method is based on measuring enzyme activity remaining after heat treatment in the absence and in the presence of ligands. The method is especially suitable for enzymes interacting with nucleosides, nucleosides and oligonucleotides since for such enzymes convenient spectrophotometric assays are available.

The Gibbs binding energy and entropy/enthalpy contributions to the interaction of calf spleen purine nucleoside phosphorylase (PNP) with the novel multisubstrate analogue DFPP-DG, as well as with DFPP-G and (S)-PMP-DAP were determined by fluorescence and calorimetric studies. Results were compared with findings for guanine - a natural reaction product and inhibitor.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.