Purification and properties of a BT-glucosidase of Hanseniaspora vineae Van der Walt and Tscheuschner with the view to its utilization in fruit aroma liberation

The β-glucosidase of Hanseniaspora vineae was purified by ion-exchange chromatography and gel filtration. Its molecular weight was 295000 PT 15000, its optimum pH was between 6 and 6–5, and its optimum temperature was 55°C. The enzyme was active against different soluble glucosides with β(1–2), β(1–3), β(1–4), β(1–6) and even aP(1–4) configurations. A glucosyltransferase activity appeared in the presence of ethanol. The enzyme was constitutive but its synthesis was repressed by glucose.     

Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes

Soluble amyloid-β peptide (Aβ) exists in the form of monomers and oligomers, and as complexes with Aβ-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Aβ(1–40) [hAβ(1–40)] from the rat brain across the blood–brain barrier. Incubation of [¹²⁵I]hAβ(1–40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [¹²⁵I]hAβ(1–40) dimer was significantly decreased by 92.7% compared with that of [¹²⁵I]hAβ(1–40) monomer. Pre-incubation with LRP-1 ligands, such as activated α2-macroglobulin (α2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [¹²⁵I]hAβ(1–40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [¹²⁵I]hAβ(1–40) monomer elimination. Purified [¹²⁵I]hAβ(1–40)/activated α2M complex and [¹²⁵I]activated α2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [¹²⁵I]activated α2M, and enhancement of [¹²⁵I]hAβ(1–40) uptake upon pre-incubation with apoE, suggesting that [¹²⁵I]activated α2M and [¹²⁵I]hAβ(1–40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAβ(1–40) from the brain across the blood–brain barrier.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.     

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis

Abstract Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Galα(1–4)βGal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Galα(1–4)βGal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A , a gene which is also required for biosynthesis and phase variable expression of the Galα(1–4)βGal LPS epitope.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.     

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC₅₀ values for blocking α4β4 and α3β4, responding to EC₂₀ concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Different GABAB-Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 µ M ) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABA_B receptors because (a) they were neither blocked by 10 µ M bicuculline nor reproduced by 10 µ M isoguvacine and (b) they were mimicked by the GABA_B agonist baclofen (3–30 µ M ), an effect fully antagonized by the GABA_B antagonist 2-hydroxysaclofen (10 µ M ). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α-, β-, and ε-PKCs). In contrast, after P21, PKC activity and α-, β-, ε-, and γ-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABA_B receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam₃CSK₄) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre-treatment of the cells with 10 μg/mL of a TLR2-specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam₃CSK₄ response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam₃CSK₄ response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ-induced tumor necrosis factor alpha production in a human monocytic cell line.     

Mitochondrial Free Radical Signal in Ceramide-Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson''s Disease

Abstract: We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by β-amyloid(Aβ) (25–35). Both Aβ(25–35)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the interleukin-1β converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH₂F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by Aβ(25–35), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. Aβ(25–35) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to Aβ(25–35) treatment and also suggest a role for calcium-regulated proteases in this process.     

Relationship of plasma steroids to germ cell development and the presence of protamine mRNA in rainbow trout during the induction of spermatogenesis with partially purified salmon gonadotropin

The gonadosomatic index (GSI) of pre–pubertal male rainbow trout, which had been injected biweekly with partially purified salmon gonadotropin (sG–G100, 50 μ mUg kg⁻¹ body weight), increased from 0.05 to 1.85 over 21 weeks from injection, while control GSI remained below 0.05. Plasma testosterone (T) increased from 2 to 11.34 ng ml⁻¹ by week 21 in injected fish, while control level remained below 1.5 ng ml⁻¹. In injected fish plasma 11–ketotestosterone (KT) and 17,20–dihydroxyprogesterone (17α20βP) levels increased from 20.2 to 41.9 and 8.9 to 219.7 ng ml⁻¹ respectively. Plasma T, 11–KT, and 17α20βP were all correlated with the GSI ( P <0–001) in injected fish. The most advanced stage of germ cells present in the control fish were spermatogonia. However, in injected fish spermatozoa were present by week 21. Eggs fertilized at this time with spermatozoa from injected fish achieved a 78% fertilization rate, whereas the testicular homogenate was incapable of fertilizing eggs.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

Polyclonal Antibodies Directed Against an Epitope Specific for the α4-Subunit of GABAA Receptors Identify a 67-kDa Protein in Rat Brain Membranes

Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α4-subunit. These anti-peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti-peptide α4 (517–523) antibodies and could then be identified by the anti-α4-antibodies as well as by the GABA_A receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABA_A receptors. Intact GABA_A receptors appeared to be retained by the immunoaffinity column because other GABA_A receptor subunit proteins like the β2/β3-subunits and the γ2-subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67-kDa protein, a 51-kDa protein could be detected by the antibody bd-28 and the anti-peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1-subunit-specific anti-peptide α1 (1–9) antibody. In contrast to the α1-subunit, the 51-kDa protein identified by the anti-α4 antibody could not be deglycosylated by N -Glycanase. The identity of the 51-kDa protein identified by the anti-α4-antibodies thus must be further investigated.     

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca²⁺ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω-conotoxin GVIA (ω-CgTX-GVIA) or ω-agatoxin IVA (ω-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca²⁺ current density of β(25–35)-treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca²⁺ uptake, which was sensitive to nimodipine. The 2',7'-dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca²⁺ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca²⁺ influx via the L-type VSCC, thereby inducing neurotoxicity.     

Glutamine Synthetase-Induced Enhancement of β-Amyloid Peptide Aβ(1–40) Neurotoxicity Accompanied by Abrogation of Fibril Formation and Aβ Fragmentation

Abstract: β-Amyloid peptide (Aβ) is the main constituent in both senile plaques and diffuse deposits in Alzheimer's diseased brains. It was previously shown that synthetic Aβs were able to form free radical species in aqueous solution and cause both oxidative damage to cell proteins and inactivation of key metabolic enzymes. We also previously demonstrated that an interaction of Aβ(1–40) with the oxidatively sensitive enzyme glutamine synthetase (GS) resulted in both inactivation of GS and an increase of Aβ toxicity to hippocampal cell cultures. In the present study the enhancement of Aβ toxicity during interaction with GS was found to be accompanied by abrogation of fibril formation and partial fragmentation of Aβ(1–40). HPLC elution profiles demonstrated the production of several peptide fragments. Analysis of the amino acid sequence of the major fragments identified them as the first 15 and the last six amino acids of Aβ(1–40). The fragmentation of Aβ was inhibited by immunoprecipitation of GS.