Maximal Binding Activity Research Articles

Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli.

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32°C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

Dynein light chain-dependent dimerization of Egalitarian is essential for maintaining oocyte fate in Drosophila

Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. We recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. In this report, we tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, we found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. We hypothesize that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity.

New Disulphide Bond in Cystatin-Based Protein Scaffold Prevents Domain-Swap-Mediated Oligomerization and Stabilizes the Functionally Active Form.

Peptide aptamers built using engineered scaffolds are a valuable alternative to monoclonal antibodies in many research applications because of their smaller size, versatility, specificity for chosen targets, and ease of production. However, inserting peptides needed for target binding may affect the aptamer structure, in turn compromising its activity. We have shown previously that a stefin A-based protein scaffold with AU1 and Myc peptide insertions (SQT-1C) spontaneously forms dimers and tetramers and that inserted loops mediate this process. In the present study, we show that SQT-1C forms tetramers by self-association of dimers and determine the kinetics of monomer–dimer and dimer–tetramer transitions. Using site-directed mutagenesis, we show that while slow domain swapping defines the rate of dimerization, conserved proline P80 is involved in the tetramerization process. We also demonstrate that the addition of a disulphide bond at the base of the engineered loop prevents domain swapping and dimer formation, also preventing subsequent tetramerization. Formation of SQT-1C oligomers compromises the presentation of inserted peptides for target molecule binding, diminishing aptamer activity; however, the introduction of the disulphide bond locking the monomeric state enables maximum specific aptamer activity, while also increasing its thermal and colloidal stability. We conclude that stabilizing scaffold proteins by adding disulphide bonds at peptide insertion sites might be a useful approach in preventing binding-epitope-driven oligomerization, enabling creation of very stable aptamers with maximum binding activity.

Melanocortin-4 Receptor in Spotted Sea Bass, Lateolabrax maculatus: Cloning, Tissue Distribution, Physiology, and Pharmacology.

Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes including energy homeostasis, reproduction, sexual function, and other functions in mammals. Recent studies suggested that teleost MC4Rs have different physiological functions and pharmacological characteristics when compared to mammalian MC4Rs. In this study, we investigated spotted sea bass (Lateolabrax maculatus) MC4R (LmMC4R) physiology and pharmacology. Spotted sea bass mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids. LmMC4R was homologous to those of several teleost MC4Rs and human MC4R (hMC4R). qRT-PCR and in situ hybridization revealed that mc4r transcripts were highly expressed in the brain, followed by pituitary and liver. Brain mc4r transcripts were down-regulated in long-term and short-term fasting challenges. LmMC4R was a functional receptor with lower maximal binding and higher basal activity than hMC4R. THIQ was not able to displace 125I-NDP-MSH but could affect intracellular cAMP accumulation, suggesting that it was an allosteric ligand for LmMC4R. In vitro studies with spotted sea bass brain cells indicated that mRNA levels of neuropeptide Y and Agouti-related peptide were down-regulated by α-MSH. In summary, we cloned spotted sea bass MC4R, and showed that it had different pharmacological properties compared to hMC4R, and potentially different functions.

SUN-480 Melanocortin-4 Receptor In Spotted Sea Bass: Cloning, Tissue Distribution, Physiology And Pharmacology

Melanocortin-4 receptor (MC4R) plays important roles in the regulation of multiple physiological processes including energy homeostasis, reproduction, sexual function and other functions in mammals. Although studies of teleost melanocortin system are less extensive, some studies revealed that teleost MC4Rs have different physiological functions and pharmacological characteristics when compared to mammalian MC4Rs. For example, mammalian MC4Rs are primarily expressed in the central nervous system whereas teleost MC4Rs are also expressed in peripheral tissues. Moreover, teleost MC4Rs have much higher basal activities compared to mammalian MC4Rs. In this study, we investigated Spotted sea bass (Lateolabrax maculatus) LmMC4R physiology and pharmacology. We identified Spotted sea bass mc4r (Lmmc4r), consisting of a 984 bp open reading frame encoding a protein of 327 amino acids. Sequence of MC4R was homologous to those of several teleost MC4Rs (over 80%) and human MC4R (hMC4R, over 70%). qRT-PCR and in situ hybridization revealed that Lmmc4r transcripts were highly expressed in brain, followed by pituitary and liver. We also revealed that brain Lmmc4r transcripts were down-regulated in the long-term and short-term fasting challenges. LmMC4R was further demonstrated to be a functional receptor by pharmacological studies. Compared to hMC4R, LmMC4R exhibited lower maximal ligand binding and higher basal activity. Endogenous ligands α-melanocyte stimulating hormone (α-MSH) and adrenocorticotropin (ACTH) could bind to LmMC4R and induce intracellular cAMP production dose-dependently. Three small molecules THIQ, Ipsen 5i and ML00253764 were not able to displace 125I-NDP-MSH but could stimulate intracellular cAMP accumulation, suggesting that they bound to LmMC4R allosterically. We isolated the brain cells of Spotted sea bass and in vitro studies indicated that gene expressions of neuropeptide Y (npy), Agouti-related peptide (agrp) and growth hormone (gh) were down-regulated when the brain cells were treated with α-MSH. In summary, we cloned Spotted sea bass MC4R, and showed that it had different pharmacological properties compared to hMC4R, and potentially different functions.

Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing.

Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP from Polaromonas JS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP from Polaromonas JS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP from Polaromonas JS666 are reported.

Determination of DNA Binding Behavior of FoxA1 Constructs Using a Gold Nanoparticle-Based High Throughput Assay

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged-helix transcription factors. It plays crucial roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. In this study, in order to determine the regions of FoxA1 necessary for efficient Deoxyribonucleic Acid (DNA) binding, we cloned, expressed and purified a series of FoxA1 constructs that contain either the DNA Binding Domain (DBD), the Transcription Activation Domain (TAD), or both. We determined the DNA binding behavior of these constructs using traditional electrophoretic mobility shift assay (EMSA) and a recently developed gold nanoparticles (AuNPs)-based fast screening method. We conclude that just the DBD region alone is not sufficient for protein-DNA binding activity. Amino acids flanking the upstream of the DBD region are required for maximal DNA binding activity. Through this study, we have also further validated the AuNPs assay for its generality and expanded the existing protocol for comparing the DNA binding behavior of multiple proteins of different charge properties and molecular weights.

Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.Methods and ResultsTo characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.Conclusions and SignificanceWe identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection

The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBSBiotin) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1J/cm2 yielded an average conjugation efficiency of ∼1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBSBiotin method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH3+ surface conjugation, NHS–Biotin, and direct physical adsorption. Taken together, the UV-NBSBiotin method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.

Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

BackgroundThe gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported.Methods and ResultsThis study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched 213SVQYHPL219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that 213SVQYHPL219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group.Conclusions and SignificanceWe identified 213SVQYHPL219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

Fine mapping of a foot-and-mouth disease virus epitope recognized by serotype-independent monoclonal antibody 4B2

VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-in-dependent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to (6)ETTLLE(11) at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif (6)ETTLLE(11) of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide (2)KKTEETTLLEDR(13) was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr(7), Thr(8), and Leu(10) are the functional residues of the 4B2 epitope Glu(6) and Leu(9) are required residues, and Glu(11) plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.

Identification of a conserved linear epitope on the VP1 protein of serotype O foot-and-mouth disease virus by neutralising monoclonal antibody 8E8

Foot-and-mouth disease virus (FMDV) serotype O remains an important threat to animal husbandry worldwide, and the variability of the virus presents a major problem for FMDV vaccine design. High-affinity neutralising antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype O and protect against future pandemics. We generated a novel monoclonal antibody (MAb) 8E8 that potently neutralised infection of FMDV O/YS/CHA/05 both in vitro and in vivo. Screening of a phage-displayed random 12-peptide library revealed that MAb 8E8 bound to phages displaying a consensus motif GDLNVRT, which is highly homologous to 146GDLQVLT 152 of the FMDV VP1 protein. Given that MAb 8E8 showed reactivity with the 146GDLQVLT 152 motif, we proposed that this motif represented a linear B-cell epitope of the VP1 protein. Western blot analysis revealed that the epitope peptide could be recognised by the positive sera from serotype O FMDV-infected pigs. The 147DLQVLT 152 motif was the minimal requirement for reactivity as demonstrated by reactivity of MAb 8E8 with several truncated peptides derived from the motif. For further mapping, a set of different extended motifs derived from the minimally reactive epitope was expressed with a GST-tag and subjected to western blot. The results showed that a 10-aa peptide 145RGDLQVLTPK 154 was the minimal unit with maximal binding activity to MAb 8E8. Subsequent alanine scanning mutagenesis studies revealed that D 147, Q 149 and V 150 are crucial for MAb 8E8 binding. Furthermore, the epitope was found to be highly conserved among different topotypes of serotype O FMDV through sequence alignment analysis and detection of MAb 8E8 for affinity to some isolates collected in China. Thus, the 8E8 epitope identified here should be helpful for designing epitope-based, intra-typic, cross-protective vaccines of serotype O FMDV.

Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

BackgroundHERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance.ResultsWild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C.ConclusionOur results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.

Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 x 10(-8) M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys106, Trp110, Tyr113, Leu114, Tyr126, Pro134, and Trp162 with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His125, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.

Involvement of AP-1 proteins in pancreatic stellate cell activation in vitro.

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis. The molecular mechanisms underlying their activation in response to profibrogenic mediators, however, are largely unknown. Extending previous studies on the transcriptional regulation of PSC activation, we have now focused on the involvement of activator protein (AP)-1. Using cultured rat PSCs, phenotypic transition of PSCs towards activated myofibroblasts was monitored by an immunoblot analysis of alpha-smooth muscle actin (alpha-SMA) expression. Transcription factor activation profiles were studied by electrophoretic mobility shift assays. DNA synthesis in PSCs was assessed through the quantification of 5-bromo-2'-deoxyuridine incorporation. Activated AP-1 complexes were detectable already before high levels of alpha-SMA were expressed. Maximal DNA binding activity of AP-1, as well as of NF-kappaB, was observed early in the course of PSC culture, while the strongest activation of STAT3 was observed much later. A detailed analysis of AP-1 complex composition revealed that phenotypic transition of PSCs towards myofibroblasts was accompanied by an increase of the JunD content relative to the one of JunB. Studies on the role of JunB and JunD in PSC activation indicated an inhibition of platelet-derived growth factor-induced DNA synthesis by antisense oligonucleotides to JunB but not JunD. The results of this study implicate AP-1 in PSC activation and suggest distinct roles of individual Jun proteins in the regulation of PSC function. In further studies, it should be analyzed whether signaling pathways involved in PSC activation might be suitable targets for antifibrotic therapies.

Effect of Active Immunization of Pony Mares against Recombinant Porcine Inhibin .ALPHA. Subunit on Ovarian Follicular Development and Plasma Steroids and Gonadotropins

Two pony mares were immunized against recombinant porcine inhibin alpha subunit three times with 39 day intervals. Clinical findings and endocrinological changes before immunization were taken as the control. The first significant rise in the anti-inhibin titre (P<0.05) in the circulation was found 27 days after the first injection. Maximum binding activity was reached by the 12th day after the second booster dose. The number of small, medium and large sized follicles had increased significantly compared to before immunization (11.75 +/- 4.30, 2.75 +/- 0.69 and 2.51 +/- 0.63 vs 6.50 +/- 1.43, 1.83 +/- 0.44 and 1.33 +/- 0.38, respectively), but the ovulation rate remained unchanged after immunization. The average plasma concentration of FSH and estradiol-17beta during the estrous cycle increased significantly (P<0.05) after immunization. These results suggest that immunization against inhibin is a useful tool to increase the number of ovarian follicles during the estrous cycle of pony mares. Moreover, the present study supported the concept that inhibin plays a major role in the control of follicular growth through its inhibitory effect on FSH secretion synergistically with steroid hormones.

Characterisation of nuclear pore complex oxalate binding protein from human kidney.

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4'-dithiocyanostilbene-2,2'-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton-high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl-Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 microM oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves.

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.

Presence of a genistein-responsive inhibitory mechanism on interleukin-1alpha-induced NF-kappaB activation.

Interleukin 1 (IL-1) is known to activate the signal transduction machinery, including the transcription factor, nuclear factor kappa B (NF-kappaB). The activation mechanism of NF-kappaB has been studied intensively, while the negative regulatory mechanisms of NF-kappaB remain to be clarified. In the present study, we found that genistein, a tyrosine kinase inhibitor, augmented IL-1alpha-dependent NF-kappaB activation, suggesting the presence of a tyrosine kinase mediating a suppression signal on NF-kappaB. As determined by luciferase reporter gene assay using kappaB-responsive element, genistein enhanced IL-1alpha-induced NF-kappaB activation. Although genistein failed to increase luciferase activity at 1 and 3 h after IL-1alpha stimulation, it induced prolonged activation beginning at 6 h after the initial stimulation. We next examined whether genistein augmented the DNA-binding activity of NF-kappaB, using electrophoretic mobility shift assay. In the case of the control experiment, the binding of NF- kappaB to the kappaB-responsive element peaked at 30 min after IL-1alpha stimulation, and decreased thereafter. In contrast, treatment with genistein maintained the maximum binding activity for at least 2 h after stimulation. Moreover, genistein enhanced the IL-1alpha-dependent degradation of IkappaBalpha. Taken together, our results indicate that genistein augments IkappaB degradation, resulting in continuous NF-kappaB activation. This suggests the possibility that tyrosine kinase negatively regulates NF-kappaB.

Structural characterization of the C2 domain of novel protein kinase Cepsilon.

Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKCepsilon-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca2+-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H2O and D2O buffers, beta sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I' band starting at 45 degrees C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm(-1) which was assigned to aggregation and a second one at 1645 cm(-1), which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKCepsilon-C2 domain has preferential affinity for PIP2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a Tm of 49 degrees C and a calorimetric denaturation enthalpy of 144.318 kJ x mol(-1). However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.