Study on the interaction between the inclusion complex of hematoxylin with β-cyclodextrin and DNA

Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(β-cyclodextrin) = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n(β-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(?)(298.15K) = 5.29 × 10? L·mol?1, and entropy worked as driven force in this action.     

Purification and characterization of ferredoxin–nitrite reductase from the eukaryotic microalga Monoraphidium braunii

Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg^–1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate K_m values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases

The role of the highly conserved C266 and L268 of pea ferredoxin–NADP⁺ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP⁺ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).     

Studies of the ferricyanide reductase activities of the mitochondrial reduced nicotinamide adenine dinucleotide-ubiquinone reductase (complex I) utilizing arylazido-β-alanyl NAD+ and arylazido-β-alanyl NADP+

The NADH and NADPH ferricyanide reductase activities present in mitochondrial NADH-CoQ reductase preparations have been studied utilizing two photoaffinity pyridine nucleotide analogues: arylazido--alanyl NAD⁺ (A3-O-{3-[N-(4-azido-2-nitrophenyl)amino]propionyl}NAD⁺) and arylazido--alanyl NADP⁺ (N3-O-{3-[N-(4-azido-3-nitrophenyl)amino]-propionyl}NADP⁺). For the NADH-K₃Fe(CN)₆ reductase activity, arylazido--alanyl NAD⁺ was found to be, in the dark, a competitive inhibitor with respect to both NADH and K₃Fe(CN)₆ withK _i,app values of 9.7 and 15.5 µM, respectively. In comparison the NADP⁺ analogue exhibited weak noncompetitive inhibitor activity for this reaction against both substrates. Upon photoirradiation arylazido--alanyl NAD⁺ inhibited NADH-K₃Fe(CN)₆ reductase up to 70% in the presence of a 25-fold molar excess of analogue over the enzyme concentration. This photodependent inhibition could be prevented by the presence, during irradiation, of the natural substrate NADH. In contrast complex kinetic results were obtained with studies of the effects of the pyridine nucleotide analogues of NADPH-K₃Fe(CN)₆ reductase activity in the dark. Photoirradiation of either analogue in the presence of the enzyme complex resulted in an activation of NADPH-dependent activity. The possibility that the NADPH-K₃Fe(CN)₆ reductase activity of complex I represents a summation of the combined ferricyanide reductase activity of the NADPH-NAD⁺ transhydrogenase and NADH oxidoreductase is suggested.     

The role of electrostatic interactions in the formation of ferredoxin–ferredoxin NADP<Superscript>+</Superscript> reductase and ferredoxin–hydrogenase complexes

A competitive Brownian model for the interaction of ferredoxin, ferredoxin NADP⁺ reductase and hydrogenase has been built. In the model, molecules of three types of proteins are placed into a cubic reaction volume, where they move under Brownian and electrostatic forces created by neighboring molecules and the solution. It has been shown that the rate of ferredoxin binding with ferredoxin NADP⁺ reductase does not change at the pH range from 5.0 to 9.0. Thus, it may be suggested that regulation of ferredoxin NADP⁺ reductase activity is mediated by other processes. On the other hand, the rate of ferredoxin binding with hydrogenase in the model depends greatly on pH: if the pH value increases from 6.0 to 8.0 the rate increases by factor of three. The increase of the pH value in the stroma under illumination results in an increase of the rate of its interaction with ferredoxin, but decreases the level of protons that are the substrate for the reaction catalyzed by the protein. Thus, the rate of hydrogen production in the chloroplast stroma is low at low pH due to the reception of a small number of electrons by hydrogenase. When the pH increases, the number of electrons that are received by the enzyme from ferredoxin also increases; thus, the rate of hydrogen production increases as well.     

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b₆f complex is active in plastoquinone reduction: implications for cyclic electron transport

In this study, we have compared three isolation methods of cytochrome b₆f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b₆f‐associated ferredoxin:NADP⁺ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH‐PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b₆f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2‐n‐nonyl‐4‐hydroxyquinoline N‐oxide, an inhibitor of the cytochrome b₆f complex at the Q_i site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c_i. The obtained data indicate that FNR associated with the cytochrome b₆f complex can participate in the cyclic electron transport as PSI‐PQ or NADPH‐PQ oxidoreductase. Moreover, we have shown that PQ can be non‐enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non‐photochemical reduction pathways of the PQ‐pool in chloroplasts.     

Over-producing soluble protein complex and validating protein–protein interaction through a new bacterial co-expression system

Many proteins exert their functions through a protein complex and protein–protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein–protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein–protein interaction of interest in a wide range of biological organisms.     

A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

Introduction

Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.

Methods

Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.

Results

TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.

Conclusions

Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.     

An acidic loop within the human soluble CD23 protein may direct the interaction between sCD23 and the αXβ2 integrin

CD23 is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor – the β₂ integrin subfamily. It has been shown that CD23 is also capable of interacting with the β₃ and β₅ integrin β-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. In this study the interaction was probed for whether or not the RKC motif governs the interaction between CD23 and the α_Xβ₂ integrin as well. This was done by performing bioinformatic docking predictions between CD23 and α_Xβ₂ integrin αI domain and SPR spectroscopy analysis of the interaction. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, in the presence of divalent metal cations the interaction showed the involvement of a novel acidic motif within the CD23 protein. This same pattern of interaction was seen in docking predictions between CD23 and the β₃I-like domain. This study thus presents an alternative site as a possible contributor to the CD23-integrin interaction exhibiting cation-dependence.     

Effect of protein modification by malondialdehyde on the interaction between the oxygen-evolving complex 33 kDa protein and photosystem II core proteins

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSII∆OEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSII∆OEE were modified, but when only OEC33 or PSII∆OEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, maximal efficiency of PSII photochemistry (F _v/F _m ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40°C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

α-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine α-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.     

Thermoregulatory differences between phenotypes in the speckled wood butterfly: hot perchers and cold patrollers?

Males of the speckled wood butterfly Pararge aegeria L. (Satyrinae), actively search for females (“patrolling”) or wait for them at particular places (“perching”). Darker males are more likely to patrol than pale ones, which are mainly territorial perchers. We studied whether this morphological variation relates to thermoregulatory differences. The relationship between thoracic temperature and ambient temperature differed between the colour types under natural conditions: darker males had on average lower body temperatures than paler males. Different activities (e.g. resting, flying) and behavioural strategies (perching or patrolling) were associated with differences in thoracic temperature: patrolling males which mainly engaged in long flights and periods of basking afterwards, had lower thoracic temperatures than perching males which engaged in very short flights, fights and basking. When resting for a while thoracic temperatures did not differ between males practising different strategies. Under laboratory conditions, darker males heated up faster than pale males but there was no difference in the thoracic temperature at which they started to fly. These results indicate that thermal requirements (or general conditions) differ between the behavioural strategies, and that behavioural differences between phenotypes (colour types) relate to differences in thermal ecology. This supports the idea that darker males are better adapted to patrolling. There is no evidence that one mate-locating strategy is always superior to the other, which coincides with the observation that both strategies co-exist. More generally, this study shows that relatively small differences in colour can have a considerable effect on thermoregulation and hence on the behavioural strategies a heliothermic insect will adopt. Received: 15 August 1997 / Accepted: 15 December 1997