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Thionins are the family of small (∼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short β-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the ‘toroidal’ pore model or the ‘carpet’ mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.

Dendritic cells (DC) are the most important antigen-presenting cells, which guide T cell activation and function, and dysregulated DC function might be one of the crucial causes of inflammatory bowel disease (IBD). It has been well-known that microbiota and their metabolites play an essential role in regulating the biology and function of DC, thus contributing to the pathogenesis of IBD. However, the underlying mechanisms remain largely unknown. Amphiregulin (AREG), a molecule of the epidermal growth factor (EGF) family, is primarily described as an epithelial cell-derived cytokine and recognized as a critical regulator of cell proliferation and tissue repair. Here, we found that DC expression of AREG depended on butyrate (a microbiota-derived short chained fatty acid), which required the interaction between butyrate and G-protein-coupled receptor 43 (GPR43). Furthermore, we found that butyrate-GPR43 interaction failed to induce AREG expression in DC deficient in B lymphocyte induced maturation protein 1 (Blimp-1). Notably, DC-derived AREG was indispensable for the protection against experimental colitis in mice. Additionally, AREG expression was significantly decreased in DC from IBD patients. Our data provide novel evidences to interpret how AREG expression is regulated in DC, and shed new light on the mechanisms whereby microbiota regulate DC function.

Sodium dodecyl sulfate (SDS), a representative anionic surfactant, is a commonly used reagent in studies of the cell membrane and cell wall. However, the mechanisms through which SDS affects cellular functions have not yet been fully examined. Thus, to gain further insights into the cellular functions and responses to SDS, we tested a haploid library of Saccharomyces cerevisiae single-gene deletion mutants to identify genes required for tolerance to SDS. After two rounds of screening, we found 730 sensitive and 77 resistant mutants. Among the sensitive mutants, mitochondrial gene expression; the mitogen-activated protein kinase signaling pathway; the metabolic pathways involved in glycoprotein, lipid, purine metabolic process, oxidative phosphorylation, cellular amino acid biosynthesis and pentose phosphate pathway were found to be enriched. Additionally, we identified a set of transcription factors related to SDS responses. Among the resistant mutants, disruption of ribosome biogenesis and translation alleviated SDS-induced cytotoxicity. Collectively, our results provided new insights into the mechanisms through which SDS regulates the cell membrane or cell wall.

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme.

Plasmin is a potent serin protease involved in a variety of biological functions, such as fibrinolysis and tissue remodeling. On performing an in vitro control assay to measure the activity of endogenous plasmin in cell lysates, a stimulatory effect of non-ionic detergent NP-40 on plasmin activity was discovered. Another non-ionic detergent, TX-100, also enhanced plasmin activity, while ionic detergents sodium deoxycholate and sodiem dodecyl sulfate abolished plasmin enzyme activity. Kinetic analysis of plasmin activity in the presence of NP-40 and TX-100 demonstrated an increase in Vmax; however, there was no change in Km values, suggesting that these detergents stimulate plasmin activity in a non-competitive manner. Fibrin plate assay indicates that NP-40 and TX-100 functionally stimulate plasmin activity by showing a dose-dependent increase in fibrinolysis.

The mechanism and detailed processes of DNA compaction and decompaction are essential for the life activities, as well as for the researches in the molecular biology, genetics and biomedicine. The compaction of two kinds of DNA molecules caused by Cetyltrimethyl Ammonium Bromide (CTAB) and their decompaction induced with sodium dodecyl sulfate (SDS) or excessive amount of CTAB have been investigated with multiple perspectives such as the UV-VIS spectrophotometry, dynamic light scattering, and zeta potential. The compaction phenomenon of DNA can easily be observed when the CTAB combines with the DNA, not just when the molar ratio QCTAB/QDNA is approximately equal to 1 as the conventional recognition, but also when QCTAB/QDNA

In common beans and lima bean, the storage protein phaseolin is difficult to degrade and SDS-resistant, a sign of kinetic stability. Kinetically stable proteins (KSPs) are characterized by having a high-energy barrier between the native and denatured states that results in very slow unfolding. Such proteins are resistant to proteolytic degradation and detergents, such as SDS. Here the method SDS-Trapping of Proteins (S-TraP) is applied directly on bean extracts to quantify the kinetic stability of phaseolin in lima bean and several common beans, including black bean, navy bean, and small red bean. The bean extracts were incubated in SDS at various temperatures (60–75 °C) for different time periods, followed by SDS-PAGE analysis at room temperature, and subsequent band quantification to determine the kinetics of phaseolin unfolding. Eyring plot analysis showed that the phaseolin from each bean has high kinetic stability, with an SDS-trapping (i.e. unfolding) half-life ranging from about 20–100 years at 24 °C and 2–7 years at 37 °C. The remarkably high kinetic stability of these phaseolin proteins is consistent with the low digestibility of common beans and lima bean, as well as their relatively high germination temperatures. From a practical perspective, this work exemplifies that S-TraP is a useful and cost-effective method for quantifying the kinetic stability of proteins in biological extracts or lysates. Depending on the protein to be studied and its abundance, S-TraP may be performed directly on the extract without need for protein purification.

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins.

The Turkevich method has been used for many years in the synthesis of gold nanoparticles. Lately, the use of plant extracts and amino acids has been reported, which is valuable in the field of biotechnology and biomedicine. The AuNPs was synthesized from the reduction of HAuCl4 3H2O by sodium glutamate and stabilized with sodium dodecyl sulfate. The optimum concentrations for sodium glutamate and sodium dodecyl sulfate in the synthesis process were determined. The characteristics of the synthesized AuNPs was analysed through UV–Vis Spectroscopy and SEM. The AuNPs have spherical shape with a mean diameter of approximately 21.62 ± 4.39 nm and is well dispersed. FTIR analysis of the AuNPs reflected that the sulfate head group of sodium dodecyl sulfate is adsorbed at the surface of the AuNPs. Thus, we report herein the synthesis of AuNPs using sodium glutamate and sodium dodecyl sulfate.

The effect of sodium dodecyl sulfate (SDS) on human, bovine, porcine, rabbit and sheep serum albumins were investigated at pH 3.5 by using various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence and dynamic light scattering (DLS). In the presence of 4.0mM SDS the secondary structure of all the albumins were not affected as measured by CD but fluorescence spectra revealed 8.0 nm blue shift in emission maxima. We further checked the stability of albumins in the absence and presence of 4.0mM SDS by urea and temperature at pH 3.5. In the absence of SDS, urea starts unfolding both secondary as well as tertiary structural elements of the all the albumins at approximately 2.0M urea but in the presence of 4.0mM SDS, urea was unable to unfold even up to 9.0M. The albumins were thermally less stable at pH 3.5 with decrease in Tm but in the presence of 4.0mM SDS, the Tm was increased. From this study, it was concluded that SDS is showing a protective effect against urea as well as thermal denaturation of albumins. This behavior may be due to electrostatic as well as the hydrophobic interaction of SDS with albumins. Further, we have proposed the mechanism of action of urea. It was found that urea interacted with proteins directly when proteins are in charged form. Indirect interaction may be taking place when the environment is more hydrophobic.

Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be induced in charged micelles at a given temperature by increasing ionic strength of the medium. We have monitored the change in organization and dynamics associated with sphere-to-rod transition of SDS micelles utilizing pyrene fluorescence. We report here, utilizing changes in the ratio of pyrene vibronic peak intensities (I(1)/I(3)), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be approximately 32. Interestingly, the apparent micellar dielectric constant exhibits a reduction with increasing NaCl concentration. The dielectric constant in rod-shaped micelles of SDS (in presence of 0.5M NaCl) turns out to be approximately 22. To the best of our knowledge, these results constitute one of the early reports on polarity estimates in rod-shaped micelles. In addition, pyrene excimer/monomer ratio shows increase in SDS micelles with increasing NaCl concentration. We interpret this increase due to an increase in average number of pyrene molecules per micelle associated with the sphere-to-rod structural transition. These results could be significant in micellar drug solubilization and delivery, and in membrane morphology changes.

Recently, the aldehyde 4-oxo-2-nonenal (ONE) was identified as a product of lipid peroxidation and found to be an effective protein modifier. In this in vitro study we investigated structural implications of the interaction between ONE and alpha-synuclein, a protein which forms intraneuronal inclusions in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Our results demonstrate that ONE induced an almost complete conversion of monomeric alpha-synuclein into 40-80 nm wide and 6-8 nm high soluble beta-sheet-rich oligomers with a molecular weight of approximately 2000 kDa. Furthermore, the ONE-induced alpha-synuclein oligomers displayed a high stability and were not sensitive to treatment with sodium dodecyl sulfate, indicating that ONE stabilized the oligomers by cross-linking individual alpha-synuclein molecules. Despite prolonged incubation the oligomers did not continue to aggregate into a fibrillar state, thus suggesting that these alpha-synuclein species were not on a fibrillogenic pathway.

The transcription factors Oct4 and Nanog are essential for the maintenance of an undifferentiated and pluripotent state in early embryonic cells, embryonic stem cells and embryonal carcinoma cells in humans and mice. These factors are co-localized to promoters of more than 300 genes, and synergistically regulate their activities. Currently, the molecular interaction between these two factors has not been well-characterized. During attempts to co-immunoprecipitate Oct4 and Nanog we found that cross-linking with dithiobis[succinimidylpropionate] was necessary to maintain their interaction. This result was supported by gel filtration analysis. Surprisingly, formaldehyde, a cross-linker commonly used during chromatin immunoprecipitation of Oct4 and Nanog, did not preserve the complex. Our findings demonstrate the effectiveness of using DSP to mitigate the instability of the interaction between these two particular proteins. Additionally, this solution may potentially allow us to identify novel members of the Oct4-Nanog complex, leading to better understanding of the regulatory mechanisms behind pluripotency.

Accumulation of misfolded Cu/Zn superoxide dismutase (SOD1) occurs in patients with a subgroup of familial amyotrophic lateral sclerosis (fALS). To identify the conversion of SOD1 from a normally soluble form to insoluble aggregates, we investigated the change of SOD1 solubility with aging in fALS-linked H46R SOD1 transgenic mice. Mutant SOD1 specifically altered to insoluble forms, which were sequentially separated into Triton X-100-insoluble/sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble/formic acid-soluble species. In spinal cords, the levels of SDS-dissociable soluble SOD1 monomers and SDS-stable soluble dimers were significantly elevated before motor dysfunction onset. In COS-7 cells expressing H46R SOD1, treatment with proteasome inhibitors recapitulated the alteration of SOD1 solubility in transgenic mice. In contrast, overexpression of Hsp70 reduced accumulation of mutant-specific insoluble SOD1. SDS-soluble low molecular weight species of H46R SOD1 may appear as early misfolded intermediates when their concentration exceeds the capacity of the proteasome and molecular chaperones.

Association of class-II phospholipase A 2 (PLA 2 ) with aggregated phospholipid substrate results in elevated levels of the Ca 2+ -dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca 2+ ion cofactor, however, in Lys49–PLA 2 homologues (Lys49–PLA 2 s), substitution of the Asp49 by Lys results in loss of Ca 2+ binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49–PLA 2 s cause Ca 2+ -independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49–PLA 2 from the venom of Bothrops jararacussu , and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca 2+ -independent membrane damaging activity by ∼4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accompanied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7–10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N -bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA 2 s.

Spinigerin is a linear antibacterial peptide derived from a termite insect. It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria, but has no hemolytic activities against human erythrocytes. In this study, we present a three-dimensional solution structure of spinigerin in SDS micelles. According to CD data spinigerin has an alpha-helical conformation in the presence of TFE, DPC micelles, and SDS micelles. The three-dimensional structure of spinigerin as determined by NMR spectroscopy contains a stable alpha-helix from Lys4 to Thr23. Spinigerin (4-21), an 18-residue fragment from Lys4 to Leu21, contains a similar content of alpha-helical structure compared to native spinigerin and was found to retain antibacterial activity, too. Therefore, this alpha-helical structure and the strong electrostatic attraction between four Lys and three Arg residues in spinigerin and the negatively charged polar head groups of the phospholipids on the membrane surface play important roles in disrupting membrane and subsequent cell death.

We describe a high-throughput procedure for measuring β-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of β-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The β-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.

Filamentous tau pathology is central to a large number of dementing disorders, including Alzheimer's disease in which polymerized tau is hyperphosphorylated. Previous studies on heparin-dependent tau polymerization, using recombinant tau isoforms lacking Cys-291, suggest that tau dimerization via Cys-322 is critical for initiation of assembly of soluble tau into filaments. We report heparin-dependent in vitro polymerization of human recombinant tau (1-383 isoform), containing both Cys-291 and Cys-322, into paired helical filaments as characterized by electron microscopy. Tau polymerization, under physiological tau concentrations in the presence of dithiothreitol (DTT), was followed by a Thioflavine S fluorescence assay. To understand the molecular basis for heparin-induced tau polymerization, we expressed and purified C291A, C322A, and C291A/C322A tau mutants. The DTT requirement for tau polymerization was abolished using either the C291A or C322A tau mutant and polymerization was not observed with the C291A/C322A tau double mutant. Analysis by sodium dodecyl sulfate gel electrophoresis showed that, unlike wild type tau, a significant amount of the C291A mutant and the C322A mutant is present as a disulfide bonded dimer. Taken together these results suggest that, in isoforms containing both Cys-291 and Cys-322, a dimeric tau with an intermolecular disulfide bond through either Cys-291 or Cys-322 is presumably acting as a seed for initiation of tau polymerization.

Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.

Metallothionein-III (MT-III) protects cerebral cortical neurons in established culture from the toxic effect of amyloid beta peptides (Abetas). Protection is concentration dependent and approaches 100% at 0.1 microM. The EC(50) value estimated at 5 microM Abeta(1-40) is 2 nM. At higher concentrations (0.1 microM), MT-III also antagonizes the trophic effect of Abeta(1-40) on cerebral cortical neurons in early cultures. Because only the fibrillar, SDS-resistant form of Abeta aggregates are thought to be neurotoxic, we analyzed and compared Abeta(1-40) aggregates formed in the presence and absence of MT-III using SDS-PAGE. Results show that aggregates formed in the absence of MT-III are predominantly SDS-resistant whereas those formed in its presence are mostly SDS-soluble. Neither MT-I nor -II exhibits any of the effects of MT-III. On the basis of these results, we propose that MT-III alleviates Abetas' neurotoxic effect by abolishing the formation of toxic aggregates of Abetas and that it may play a specific and important role in protecting the brain from the deleterious effects of Abetas.

The in vitro transcription of glnL, initiated at the gene's specific promoter, was stimulated by adding a proteinaceous fraction purified from a culture at 42 degrees C, while the corresponding fraction from a culture at 28 degrees C was not stimulating. The stimulating fraction was not effective on in vitro transcription of some other genes. These stimulating and nonstimulating fractions were further purified and comparatively analyzed in sodium dodecyl sulfate polyacrylamide gel electrophoresis, which revealed that the amounts of 84 kD protein had predominantly increased in the stimulating fraction, but not in the nonstimulating fraction.

The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between -170 and -150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60 degrees C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.

The 26S proteasome is assembled from the 20S proteasome and the regulatory subunit complex in an ATP-dependent manner. In the present study, we found that the ATP-dependent activity and the protein amount of the 26S proteasome change during growth of the budding yeast Saccharomyces cerevisiae.Both levels in the stationary phase are higher than those in the exponentially growing phase. On the other hand, the levels of the 20S proteasome appear to remain unchanged during growth. These results suggest that the 26S proteasome undergoes a growth-dependent change and that the 26S proteasome plays a role in the survival of yeast cells under starvation conditions.

Rational modification of an existing cationic alpha-helical antimicrobial peptide (ESF1) for improved activity by increasing amphipathicity was undertaken. ESF1 and two variants (GR7 and SA3) were synthesized and tested for activity range, minimum inhibitory concentration, and hemolytic activity. Biological activity was related to structure as determined by circular dichroism. The substitution of arginine for glycine in position seven was found to increase antimicrobial activity without effecting hemolysis. Increased activity was related to stronger alpha-helix formation in buffer. Increased beta-sheet formation in micellar SDS was observed and speculated to be due to a stronger ability of the variants to form multimolecule complexes, a feature consistent with existing models of cationic peptide activity.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 8 (PI8) is a 45 kDa member of the ovalbumin family of serpins that is an inhibitor of trypsin-like proteinases through the use of Arg 339 as the inhibitory P 1 amino acid residue in its reactive site loop. In this study, we have described the inhibitory mechanism of recombinant human PI8 towards chymotrypsin. PI8 formed an SDS-stable complex with and inhibited the amidolytic activity of chymotrypsin via a two-step mechanism with an overall equilibrium inhibition constant of 1.7 nM and an overall second-order association rate constant of 1.0 × 10 4 M −1 s −1 , utilizing Ser 341 as the P 1 residue. The use of separate reactive site loop residues by PI8 to inhibit distinctly different classes of proteinases not only supports the hypothesis of the existence of the serpin reactive site as a highly mobile and flexible loop, but also suggests an evolved function in which separate amino acid residues can be used to broaden the inhibitory specificity of PI8.

Insulin-like growth factor binding proteins (IGFBPs) are important modulators of IGF actions. IGFBP-3 and IGFBP-5 can bind to the extracellular matrix of a number of cell types. We now describe a new posttranslational structural modification of IGFBP-3 and IGFBP-5, which could play a role in determining their localization. We incubated radioiodinated forms of all six IGFBPs in the presence of a redox buffer consisting of 10 mM reduced glutathione and 0.2 mM oxidized glutathione. Under these conditions IGFBP-3 and IGFBP-5, but not the other IGFBPs, formed high molecular weight disulfide-linked multimers. Heparin and a peptide encompassing the high-affinity heparin-binding site in the C-terminal portion of IGFBP-3 were capable of blocking the multimerization of IGFBP-3. IGFBP-3, but not IGFBP-1, was shown to be able to self-associate non-covalently, which could be a requisite first step in the formation of covalent multimers. The self-association of IGFBP-3 required the high-affinity heparin-binding site in the C-terminal portion of the molecule.

Pathogenic mycobacteria are generally slow growing organisms and it takes several weeks to evaluate inhibitors of growth. Therefore, for rapid screening of the inhibitors of mycobacterial growth, a beta-galactosidase reporter system has been described which utilises a recombinant Mycobacterium smegmatis mc(2)155 expressing E. coli lacZ gene as the test organism. The assay is based on production of beta-galactosidase in presence of drugs during growth. A correlation between beta-galactosidase production and colony forming ability of mycobacteria was obtained. beta-galactosidase production was inhibited within 6 h by front line standard antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, pyrazinamide and ofloxacin at their reported MICs. The assay was performed on mycobacterial cells permeabilized with chloroform and sodium dodecyl sulfate.

Cytochrome b558 is a component of the superoxide-generating system in neutrophils and plays key roles in both the assembly of a functional complex with cytosolic proteins and shuttling an electron from NADPH to molecular oxygen. To determine the role of predicted hydrophilic domains of gp91-phox, a glycosylated subunit of the cytochrome, we synthesized peptides corresponding to the regions and tested whether they affected superoxide generation in the cell-free system obtained from human neutrophils. Among twelve peptides tested, six peptides, four of which correspond to previously unreported regions, inhibited superoxide generation in the cell-free system. All of the active peptides were effective when added to the system before activation with sodium dodecyl sulfate. Four peptides, including two peptides corresponding to two newly identified regions, inhibited the translocation of the cytosolic components, p47-phox and p67-phox. The extent of inhibition on translocation of these components varied depending on the peptide used.

Periodic acid-Schiff-positive colloids occur in the pituitary glands of many vertebrates. These colloids are surrounded by folliculo-stellate (FS) cells and increase dramatically in number and size with age. The characterization of these colloidal substances is necessary for the establishment of FS cell function. In a previous study we showed that these colloids in the senescent pituitary contain a number of glycoproteins; however, the characterization of these glycoproteins has not yet been carried out. To characterize the pituitary colloid, we purified the colloids by centrifugation and Percoll gradient. The isolated colloids were unable to be solubilized in a regular buffer since they were very dense. However, solubilization was accomplished in sodium dodecyl sulfate (SDS) lysis buffer following treatment with exoglycosidase. Analysis using SDS-polyacrylamide gel electrophoresis showed that the porcine pituitary colloids contain four proteins, CP26, CP40, CP48 and CP60, whose estimated molecular weights were 26 kD, 36-43 kD, 48 kD, and 60 kD, respectively. Staining with concanavalin A (ConA) showed that both CP26 and CP40 were glycoproteins. Moreover, N-terminal amino acid sequencing and Western blot analysis revealed CP26, CP48 and CP60 to be porcine albumin while CP40, the major protein in the colloids, proved to be clusterin. Therefore we report here that the glycoproteins that occur in senescent porcine pituitary colloids are of two types, namely, albumin fragments and clusterin.

We found that high molecular polyanions (PAs) such as dextran sulfate and heparin burst some damaged or dead lymphocytes in lymphocyte suspension isolated from living bodies, and clarified morphologically the mechanism involved in this. PAs passed through damaged cell membranes and reached the chromatin in the nucleus. Electron micrographs suggested that they then extracted histones from the chromatin and formed complexes with them, and that freed DNA formed a gel binding with the complexes in the nucleus, and the nucleus became swollen and burst due to the gel swelling pressure. To support this conjecture, the nuclei of lymphocytes were stained with propidium iodide and exposed to light to stabilize them. The stabilized nuclei were not swollen after the addition of PAs and the lymphocytes did not burst. The lymphocyte bursting effect of PAs was attributed to their negative charges which could not destroy intact cells. At this point, PAs are different from sodium dodecyl sulfate which destroys intact cells completely.

We have tried to refold thermally-denatured MM-CK using detergent and cyclodextrins as protein folding assistants. This procedure, named artificial chaperone-assisted refolding, has been extensively tested to refold carbonic anhydrase B. Here, we describe a study which shows that this procedure can be applied to refold a dimeric multidomain protein : MM-creatine kinase. The pair SDS/hydroxy-propyl beta-cyclodextrin was used in this sequential refolding method. In the first step, the protein was denatured by SDS which is able to strongly inhibit aggregation. In the second step, hydroxy-propyl beta-cyclodextrin, an efficient SDS-stripping agent, is added and the denatured enzyme can regain its native structure as shown by the 75% reactivation. In conclusion, this study suggests that this procedure can be widely used to refold monomeric, as well as oligomeric, multidomain proteins.

Locusta adipokinetic hormone-III (AKH-III, < QLNFTPWWa) and three analogues have been studied by CD spectroscopy. The AKH peptides examined in which the tryptophan residues are adjacent present a distinctive negative-positive CD signature, in aqueous solution, that increases at low temperatures in ethanediol/water. In the presence of 0.6% SDS, a positive-minus CD is observed. The separation of the two tryptophan residues by an aliphatic amino acid results in a CD, in aqueous solution inverted from negative to positive CD at approximately 225nm. For the peptides with two adjacent tryptophan residues, the bisignate nature of this tryptophan-based CD at lower temperatures or in SDS indicates that the indole/indole orientation can adopt two limiting conformations. There is a correlation between the size of the negative CD at 225 nm and the AKH peptide potency which may indicate a preferred indole/indole orientation by the receptor.

Transglutaminase was purified to homogeneity from leaves of soybean ( Glycine max ). The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 80,000 daltons. This purified enzyme catalyzed the incorporation of [ 14 C]-putrescine into N,N′-dimethylcasein as a protein substrate. With N,N′-dimethylcasein, the Km values for putrescine, spermidine and spermine were 109, 42 and 69 μM, respectively. The optimum pH and temperature for the enzyme reaction were 7.6 and 37°C. Ca 2+ was not an absolute requirement for enzyme activity unlike animal transglutaminases. The enzyme was activated by dithiothreitol, but inhibited by GTP. With molecular weight of this enzyme, this inhibition of enzyme activity by GTP indicates that this enzyme is very similar to known tissue transglutaminases in animals.

The effect of the S-nitrosothiol (RSNO) on the activation of NADPH oxidase in human neutrophils was studied using an in vitro translocation system in which an anionic amphiphil, such as sodium dodecyl sulfate or arachidonate, plays a role as an activator. When membranes pretreated with RSNO and a cytosol fraction from resting neutrophils were combined to reconstitute the NADPH oxidase, both translocation of the cytosolic NADPH oxidase components such as p47 phox and p67 phox to the plasma membrane fraction and subsequent superoxide generation was inhibited. However, RSNO had no effect on O 2 − production when added after enzyme activation. A similar inhibition of translocation of recombinant p47 phox was observed with RSNO-treated membrane. When the RSNO-treated membrane fraction was exposed to 2-mercaptoethanol the inhibition was reversed. The data suggest that RSNO inhibits translocation of p47 phox or p47 phox containing cytosolic complex via a direct effect on the membrane component of the NADPH oxidase.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met 169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate Either ADP or adenosine 5′-(β,γ-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Activation of superoxide-generating NADPH oxidase system of human neutrophils involves phosphorylation-dependent translocation of p47phox and other cytosolic components to the plasma membrane. In contrast to the stimulation of the NADPH oxidase in intact cells, however, the activation of cell-free system requires the addition of anionic amphiphiles such as sodium dodecyl sulfate (SDS) and arachidonate. In this system, translocation of p47phox is also an essential step for activation, but phosphorylation is not required. The basis of this difference in oxidase activation is not yet clear. We now report that in a cell-free oxidase system, phosphorylated recombinant p47phox can be translocated to the membrane in the absence of SDS or arachidonate. These findings suggest that both phosphorylation and SDS could cause a common change in conformation or charge of p47phox that may result in the association of p47phox with the plasma membrane.

IRE1α (Inositol-requiring enzyme 1 α), an endoplasmic reticulum (ER)-resident sensor for mammalian unfolded protein response, is a type I transmembrane protein which has a bifunctional enzyme containing kinase and RNase domains. Although the luminal domain and cytosolic domain of IRE1α are thought to play crucial roles in regulating the protein activity, no functional and structural studies of the transmembrane domain exist thus far. Herein, using CD spectroscopy, we report that the transmembrane domain of the IRE1α is alpha-helical in a membrane-like environment. In addition, SDS-PAGE and FRET analyses support that the transmembrane domain forms oligomers in SDS micelles. Thus, the study would provide insights into how the transmembrane domain plays a role in regulating the IRE1α protein activity.

LPS is the most important antigen of Brucella bacteria which are gram-negative facultative intracellular pathogens infecting a large proportion of animals and humans in the world. In order to get insights into the immune response mechanisms monitored by Brucella , its LPS was used as a model antigen. S-LPS, R-LPS, lipid A and O-chain purified from Brucella abortus were tested in their capacity of inducing SDS-resistant MHC class II molecules after incubation with murine B lymphoma cells. S-LPS and O-chain gave a significant response sugesting that O-chain might induce an association with class II itself or might act as a carrier for antigens to bind MHC class II molecules.

Kinetic analyses indicate that the inhibitory effects of the nonionic detergents Triton X-100 and Nonidet P-40 on chloramphenicol acetyltransferase are exerted by a competitive and a non-competitive mechanism with respect to the substrates chloramphenicol and acetyl-CoA, respectively. Comparison with nonionic detergents without an aromatic moiety like that present in Triton X-100 and Nonidet P-40 suggests that the aromatic groups in these two detergents may compete with chloramphenicol for binding to the hydrophobic, active site in the chloramphenicol acetyltransferase.

A cellobiohydrolase (CBH3) of Clostridium thermocellum was isolated from the recombinant strains of Escherichia coli. The enzyme was shown to have a Mr of 78 kDa and isoelectric point of 4.75. The hydrophilic nature of the enzyme was confirmed by its unretarded elution on gel filtration column. The enzyme had a binding affinity for the microcrystalline substrate (Avicel). The pH and temperature optimum were determined as 6.5 and 60 degrees C. The enzyme was found to be active on p-nitrophenyl cellobioside, carboxymethyl cellulose, Avicel, amorphous cellulose, lichenan and xylan. The most distinguishing feature of CBH3 was the degradation of a variety of natural crystalline substrates, with maximum activity on filter paper. The enzyme was tolerant to high levels of cellobiose, susceptible to heavy metals and was protected by DTT and calcium at higher temperatures. This is the first report of a highly active cellobiohydrolase produced in C. thermocellum.

A novel Ca 2+ -binding protein which is termed S-100P was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-100P. Isoelectric point of S-100P was pI=4.6. S-100P did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45 Ca overlay technique. A ninety amino acid sequence of S-100P was determined. It is 49% identical with human S-100β, 38% with human calcyclin, and 37% with human cystic fibrosis antigen.

The lubricating properties of human submandibular-sublingual salivary fractions were examined using a servohydraulic model of mandibular movement. Fractions containing statherin exhibited a strong tendency to boundary lubrication. The lubricity of purified statherin was confirmed and compared to the amphipathic molecules gramacidin S and sodium dodecyl sulfate. Contact angle measurements of statherin paralleled the other amphipathic molecules. The helical content of statherin increased in trifluoroethanol indicating the presence of amphipathic helical regions. CD studies and hydrophobic moment calculations indicated that statherin adopts an amphipathic helical conformation at the N-terminus. An energy-minimized model of the polar N-terminal residues 1-15 suggested that this domain could be positioned in space to interact with a hydroxyapatite substrate. These data imply that under appropriate conditions statherin may display an amphipathic nature which enables it to function as a boundary lubricant on enamel.

The phenoloxidase system, which is involved in encapsulation and melanization of foreign objects in crustacean, is found to be present in an inactive proenzyme form in the hemocytes of the lobster, Homarus americanas. Activation of the enzyme could be achieved either by treatment with an anionic detergent such as sodium dodecyl sulfate, or by a cationic detergent such as cetylpyridinium chloride, but not by either nonionic detergent or zwitterionic detergent. In addition, a number of fatty acids also activated the proenzyme. However, phospholipids, especially lysolecithin proved to be the most potent activator of prophenoloxidase. Therefore, it is proposed that apart from the well established proteolytic mode of activation, prophenoloxidase can also be activated by this alternative mode involving lipids.

In the present work we show the existence of two Na+/K(+)-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca2+ sensitivities. The catalytic subunits (alpha 1, alpha 2) of Na+/K(+)-ATPase were labelled by fluorescein-isothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca2(+)-sensitivities were studied by comparing the kinetics of Ca2+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabain-sensitive part (alpha 2 type) of Na+/K(+)-ATPase enzyme could only be inhibited by micromolar Ca2+. Furthermore, treatment of the microsomal preparation with 1mM N-ethylmaleimide selectively inactivated the high Ca2+ sensitive isoform of myometrial Na+/K(+)-ATPase.

Using a non-denaturing digitonin-based polyacrylamide gradient gel electrophoretic system we identified the dihydropyridine-sensitive Ca2+ channel from skeletal muscle as a high molecular weight protein of greater than 700 kDa. When this protein was excised from the native gels and re-electrophoresed into SDS gels, it dissociated into the alpha 1, alpha 2, beta, gamma and delta peptides previously suggested to be putative subunits of these Ca2+ channels. The stoichiometry of the alpha 1:alpha 2:beta:gamma peptides was (-)1:1:1:1. The presence of the alpha 1 and alpha 2 peptides in the high molecular weight native complex was directly demonstrated with anti-alpha 1 and anti-alpha 2 antibodies. The apparent specific association of the peptides was demonstrated by the finding that the previously separated alpha 1 and alpha 2 peptides did not co-migrate with the native complex in non-denaturing gels. The results of this previously untried analysis support the concept that the skeletal muscle Ca2+ channels are multisubunit proteins. The combined non-denaturing and denaturing gel analyses may be of general utility for the analysis of other membrane proteins.

The structure of the Ah receptor previously has been extensively characterized by reversible binding of the high affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report the use of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as a photoaffinity ligand for Ah receptor from the mouse hepatoma cell line Hepa-1c1c9. Both cytosolic and nuclear forms of Ah receptor could be specifically photoaffinity-labeled, which allowed determination of molecular mass for the two forms under denaturing conditions. After analysis by fluorography of polyacrylamide gels run in the presence of sodium dodecyl sulfate, molecular mass for the cytosolic form of Ah receptor was estimated at 92,000 ± 4,300 and that for the nuclear form was estimated at 93,500 ± 3,400. Receptor in mixture of cytosol and nuclear extract (each labeled separately with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin) migrated as a single band. These results are consistent with the presence of a common ligand-binding subunit of identical molecular mass in both cytosolic and nuclear complexes.