Presence Of Nonionic Detergent Research Articles

Purification of a Thermostable β-mannanase from Paenibacillus Thiaminolyticus - characterization and its Potential Use as a Detergent Additive

Endo-1, 4- β- D-mannanase (EC 3.2.1.78) is a glycoside hydrolase involved in random cleavage of β-1, 4- D-manno-pyranosyl linkages within mannans and heteromannans and generates branched and linear oligosaccharides. A β-mannanase was purified from a thermotolerant bacterium Paenibacillus thiaminolyticus isolated from a soil sample. Enzyme was purified to homogeneity with specific activity of 8812 U/mg protein. Sodium dodecyl sulfate (SDS) and native poly-acryl amide gel electrophoresis indicated that the purified mannanase is a monomeric protein with a molecular mass of 38 kDa. The purified enzyme was found to be maximally active at temperature and pH of 60°C and 7.0, respectively. It was stable at 55°C for 24 h and maintained more than 50 % activity up to 3 h at 60°C. The enzyme was very stable in the pH range of 5.0-9.0. Purified β-mannanase demonstrated high stability after 1 h of pre-incubation with most of the tested organic solvents. Enzyme retained significant stability in the presence of various detergent additives, commercially available detergents and dish washing liquids. The high compatibility and substantial stability in the presence of nonionic detergents and dishwashing liquids confirmed its utility as an additive to dish washing liquids and laundry detergents. Enzyme exhibited efficacious de-staining of heteromannan based stains of chocolate ice cream and salad dressing in the wash performance test for detergent application. It also exhibited anti-soil redeposition effect on cotton swatches treated with tennis court clay and heteromannans.

Potential of bacteriophages as disinfectants to control of Staphylococcus aureus biofilms

BackgroundStaphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms.ResultsSDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 109 CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ.ConclusionsOur results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Superstructure formation by RodZ hexamers of Shigella sonneimaintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms “superstructures” of high molecular mass that dissociate into a midsized “basal complex” in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

Methods for Extraction of RNA, Proteins, or Protein Complexes from Subcellular Compartments of Eukaryotic Cells.

Eukaryotic organelles or subcellular compartments can be selectively isolated based on their physical density and their stability in the presence of nonionic detergents. This chapter describes a protocol for the preparation of cytoplasm, nucleoplasm, and chromatin, in addition to isolation of RNA and proteins from these fractions. Proteins and protein complexes stably associated with chromatin and other high molecular weight nuclear components can be extracted under non-denaturing conditions by enzymatic digestion of RNA and DNA. The chapter also includes a detailed extraction protocol from highly purified nuclei.

Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

Understanding Colicin N Import into Gram Negative Bacterial Cells using Small Angle Neutron Scattering

Escherichia coli (E. coli) are gram-negative bacteria commonly found in human guts but which can cause food poisoning or severe systemic infections such as meningitis. Unlike other bacteria, gram-negative cells have an additional asymmetric outer membrane (OM) which consists of tightly-packed lipopolysaccharides in the outer leaflet of membrane and phospholipids on the inside. The OM is a selective permeability barrier and protects E. coli from antibiotics and bile acids. The occurrence of antibiotic-resistant bacteria is becoming increasingly serious and owing to the tightly-packed OM gram-negative bacteria can show even higher antibiotic resistance. In the search for novel effective antibiotics, Colicin N (ColN) is a promising model. ColN is a bacterial toxin produced and secreted by E. coli in time of stress. It translocates across the OM of target cells and kills via a voltage dependent channel in the inner membrane. ColN exploits outer membrane protein F (OmpF) as both a receptor and translocator. The pore-forming domain binds to the outside of OmpF (1, 2). Here we study the OmpF/ColN translocation complex in the presence of non-ionic detergents, where binding is driven by the translocation domain. The structure of complexes was determined by small angle neutron scattering using the SANS2D beam line and utilizing a contrast variation strategy with selective deuteration of proteins and Octyl Glucoside (OG). Mixing of three OG forms achieved exact contrast matches with the solvent. This enabled a low resolution structure of the translocon to be derived from ab initio modeling and revealed that the translocation and receptor-binding domains of ColN bind to the middle of the OmpF trimer.1. Baboolal, T. G. et al. Structure16, 371, (2008).2. Clifton, L. A. et al. J. Biol. Chem.287, 337,(2012).

Observations of immuno-gold conjugates on influenza viruses using waveguide-mode sensors.

Gold nanoparticles were conjugated to an antibody (immuno-AuNP) against A/Udorn/307/1972 (H3N2) influenza virus to detect viruses on a sensing plate designed for an evanescent field-coupled waveguide-mode sensor. Experiments were conducted using human influenza A/H3N2 strains, and immuno-AuNP could detect 8×105 PFU/ml (40 pg/µl) intact A/Udorn/307/1972 and 120 pg/µl A/Brisbane/10/2007. Furthermore, increased signal magnitude was achieved in the presence of non-ionic detergent, as the virtual detection level was increased to 8×104 PFU/ml A/Udorn/307/1972. Immuno-AuNPs were then complexed with viruses to permit direct observation, and they formed a ring of confined nanodots on the membrane of both intact and detergent-treated viruses as directly visualized by scanning electron microscopy. With this complex the detection limit was improved further to 8×103 PFU/ml on anti-rabbit IgG immobilized sensing plate. These strategies introduce methods for observing trapped intact viruses on the sensing plates generated for optical systems.

Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

Optimal Conditions for Continuous Immobilization of Pseudozyma hubeiensis (Strain HB85A) Lipase by Adsorption in a Packed-Bed Reactor by Response Surface Methodology

This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150 min incubation time, pH 4.76, and an enzyme/support ratio of 1282 U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone.

Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

BackgroundNonstructural glycoprotein 4 (NSP4) encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types.MethodsNSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay.ResultsSecreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane.ConclusionOur study is the first to analyze an authentic (i.e. non-recombinant) form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain, secreted NSP4 remains soluble in an aqueous environment as an oligomeric lipoprotein that can bind to various cell types via an interaction with glycosaminoglycans. This broad cellular tropism exhibited by NSP4 may have implications for the pathophysiology of rotavirus disease.

A two-component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram-negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue. F. novicida KdtA is bi-functional, but F. novicida contains a membrane-bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co-transcribed genes. KdoH1 (related to sialidases) has a single predicted N-terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non-ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose-deficient E. coli mutant that makes Kdo(2) -lipid A as its sole LPS, accumulates Kdo-lipid A when expressing the both hydrolase components, and 1-dephospho-Kdo-lipid A when expressing both the hydrolase and the Francisella lipid A 1-phosphatase (LpxE).

Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat

Abstract Twenty-eight organic solvent tolerant bacteria were isolated from crude oil contaminated samples, out of which, AK1871 isolate produced a solvent, detergent and oxidizing agent tolerant serine alkaline protease. Based on the morphological and biochemical characteristics, FAME analysis as well as 16S rRNA gene sequence, the isolate is identified as Bacillus cereus . A 58-fold purification of protease was achieved by a three-step purification procedure. This protease is active over a broad range of pH (6.0–9.0, optimum at 8.0); and temperature from 40 °C to 70 °C (optimum at 60 °C). Li + , Ba 2+ , K + , Mg 2+ and Mn 2+ did not affect, while heavy metals like Cr 3+ , Hg 2+ and Cu 2+ inactivated the enzyme. It is stable in the presence of non-ionic detergents (Triton X-100 and Tween 80), and oxidizing and bleaching agents (hydrogen peroxide). The protease exhibited noteworthy stability and activation in the presence of organic solvents with log P values equal to or more than 2.0. This protease could be used in detergent formulations, enzymatic peptide synthesis, biotransformation reactions and in the formulation of antifouling agent. This is the first report on the study of organic solvent tolerant protease from a marine organic solvent tolerant bacterium isolated from the Gulf of Khambhat.

Effects of Detergents on Activity, Thermostability and Aggregation of Two Alkalithermophilic Lipases from Thermosyntropha lipolytica

Thermosyntropha lipolytica DSM 11003, an anaerobic thermophilic lipolytic bacterium, produces the two alkalithermophilic lipases, LipA and LipB. Among all tested detergents, the two lipases were mostly affected by SDS when used at concentrations below its critical micelle concentration (CMC). In the absence of SDS, the vmax of both LipA and LipB were 12.6 U·mg-1 and 13.3 U·mg-1 and K0.5 were 1.8 mM and 1.65 mM, respectively at 96°C and pHopt25ºCof 9.4-9.6. In the presence of 0.2% SDS, the vmax increased to 105 U·mg-1 and 112 U·mg-1, and K0.5 values decreased to 200 µM and 140 µM for LipA and LipB, respectively. Inhibitory assays of lipases using diisopropyl p-nitrophenylphosphate (E600) with increasing concentration of SDS and Tween 20 strongly suggest that SDS and Tween 20 do bind to the lid domain and/or active site pocket, thus promoting conformational changes that facilitate active site accessibility for the substrate. The two lipases exhibited moderate activation in the presence of nonionic detergents when used below their CMC values. Both lipases were found to exhibit strong tendency to aggregate as observed through gel filtration chromatography and gradient native gel electrophoresis. The addition of 1.0% (w/v) SDS led to disaggregation as the lipases were eluted corresponding to their monomeric mass (based on SDS gel electrophoresis value) and caused a significant decrease in thermostability, suggesting that, enzyme aggregation might be a major contributor to the high thermostability of LipA and LipB.

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module: MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of approximately 70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.

Use of a Bacteriophage Lysin, PlyC, as an Enzyme Disinfectant againstStreptococcus equi

Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, PlyC, as a narrow-spectrum disinfectant against S. equi. This enzyme was active against >20 clinical isolates of S. equi, including both S. equi subsp. equi and S. equi subsp. zooepidemicus. Significantly, PlyC was 1,000 times more active on a per weight basis than Virkon-S, a common disinfecting agent, with 1 microg of enzyme able to sterilize a 10(8) CFU/ml culture of S. equi in 30 min. PlyC was subjected to a standard battery of tests including the Use Dilution Method for Testing Disinfectants and the Germicidal Spray Products Test. Results indicate that aerosolized PlyC can eradicate or significantly reduce the S. equi load on a variety of materials found on common stable and horse-related equipment. Additionally, PlyC was shown to retain full activity under conditions that mimic a horse stable, i.e., in the presence of nonionic detergents, hard water, or organic materials. We propose PlyC as the first protein-based, narrow-spectrum disinfectant against S. equi, which may augment or supplement the use of broad-spectrum disinfectants in barns and stables where equine strangles is prevalent.

Cdk5–p39 is a labile complex with the similar substrate specificity to Cdk5–p35

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.

Enhanced Macrocyclizing Activity of the Thioesterase from Tyrocidine Synthetase in Presence of Nonionic Detergent

Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.

Intracellular domains of the δ-subunits of Torpedo and rat acetylcholine receptors—expression, purification, and characterization

There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the δ-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His) 6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni–NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular δ-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.

Killer toxin of Pichia membranifaciens and its possible use as a biocontrol agent against grey mould disease of grapevine.

The use of Pichia membranifaciens CYC 1106 killer toxin against Botrytis cinerea was investigated. This strain exerted a broad-specificity killing action against other yeasts and fungi. At pH 4, optimal killer activity was observed at temperatures up to 20 degrees C. At 25 degrees C the toxic effect was reduced to 70%. The killer activity was higher in acidic medium. Above about pH 4.5 activity decreased sharply and was barely noticeable at pH 6. The killer toxin protein from P. membranifaciens CYC 1106 was purified to electrophoretic homogeneity. SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa. Killer toxin production was stimulated in the presence of non-ionic detergents. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain of B. cinerea. The symptoms of infection and grey mould observed in Vitis vinifera plants treated with B. cinerea were prevented in the presence of purified P. membranifaciens killer toxin. The results obtained suggest that P. membranifaciens CYC 1106 killer toxin is of potential use in the biocontrol of B. cinerea.