Zinc-induced Alzheimer's Aβ1–40 Aggregation Is Mediated by Conformational Factors
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26464
ISSN1083-351X
AutoresXudong Huang, Craig Atwood, Robert D. Moir, Mariana A. Hartshorn, Jean‐Paul Vonsattel, Rudolph E. Tanzi, Ashley I. Bush,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe heterogeneous precipitates of Aβ that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Aβ1–40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Aβ is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Aβ is mediated by a peptide dimer and favored by conditions that promote α-helical and diminish β-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behaviorin vitro. Zinc-induced Aβ aggregation was dependent upon the presence of NaCl, was enhanced by α-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Aβ aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Aβ aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Aβ interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Aβ assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Aβ deposit in the disease. The heterogeneous precipitates of Aβ that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Aβ1–40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Aβ is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Aβ is mediated by a peptide dimer and favored by conditions that promote α-helical and diminish β-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behaviorin vitro. Zinc-induced Aβ aggregation was dependent upon the presence of NaCl, was enhanced by α-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Aβ aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Aβ aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Aβ interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Aβ assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Aβ deposit in the disease. The pathological hallmark of Alzheimer's disease is the abundant accumulation in the brain of Aβ, a 39–43-amino acid peptide, as morphologically heterogeneous deposits in the neuropil (senile plaques) and cerebral blood vessels (congophilic angiopathy) (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4379) Google Scholar, 2Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3791) Google Scholar). 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We have recently reported that Aβ itself specifically and saturably binds zinc, manifesting high affinity binding (K D = 107 nm) with a 1:1 (zinc:Aβ) stoichiometry and low affinity binding (K D = 5.2 μm) with a 2:1 stoichiometry (16Bush A.I. Pettingell Jr., W.H. Paradis M. Tanzi R.E. J. Biol. Chem. 1994; 269: 12152-12158Abstract Full Text PDF PubMed Google Scholar). This binding is probably histidine-mediated since it is abolished by acidic pH (no binding at pH 6). The zinc-binding site was mapped to a stretch of contiguous residues between positions 6–28 of the Aβ sequence. Occupation of the zinc-binding site, which straddles the lysine 16 position of α-secretase cleavage (17Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1215) Google Scholar, 18Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Crossref PubMed Scopus (749) Google Scholar), inhibits α-secretase type (tryptic) cleavage, and so may influence the generation of Aβ from amyloid protein precursor (APP) and may increase the biological half-life of Aβ by protecting the peptide from proteolytic attack (16Bush A.I. Pettingell Jr., W.H. Paradis M. Tanzi R.E. J. Biol. Chem. 1994; 269: 12152-12158Abstract Full Text PDF PubMed Google Scholar). We also found that concentrations of zinc ≥1 μm rapidly destabilize human Aβ1–40 solutions, inducing rapid Aβ precipitation (19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar) that is highly specific for zinc, although both copper(II) and iron(II) can induce partial aggregation at equivalent concentrations (20Bush A.I. Moir R.D. Rosenkranz K.M. Tanzi R.E. Science. 1995; 268: 1921-1923Crossref PubMed Scopus (32) Google Scholar). Meanwhile, rat Aβ1–40 (with substitutions of Arg → Gly, Tyr → Phe, and His → Arg at positions 5, 10, and 13, respectively) binds zinc less avidly (K a = 3.8 μm, with 1:1 stoichiometry) and is unaffected by zinc at these concentrations, perhaps explaining the scarcity with which these animals form cerebral Aβ amyloid (21Johnstone E.M. Chaney M.O. Norris F.H. Pascual R. Little S.P. Brain Res. Mol. Brain Res. 1991; 10: 299-305Crossref PubMed Scopus (305) Google Scholar, 22Shivers B.D. Hilbich C. Multhaup G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (402) Google Scholar). In the absence of zinc, the solubilities of the rat and the human Aβ species are indistinguishable (19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar). We observed that iodinating the peptide on the 10th residue of tyrosine attenuated zinc-mediated precipitation (19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar), and since this residue is substituted with a phenylalanine in the rat species, we concluded that this tyrosine is critical in coordinating zinc to the human peptide. These observations are important because zinc is abundant in the same neocortical regions where Aβ deposits are most commonly found, and high micromolar zinc concentrations are achieved during glutamatergic neurotransmission (24Assaf S.Y. Chung S.-H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1046) Google Scholar, 25Howell G.A. Welch M.G. Frederickson C.J. Nature. 1984; 308: 736-738Crossref PubMed Scopus (713) Google Scholar), suggesting an explanation for the propensity of Aβ to deposit close to the neocortical synaptic vicinity. Recently, we have also found evidence that zinc mediates the assembly of a significant fraction of Aβ deposits in Alzheimer-affected postmortem brain tissue. 1R. A. Cherny, K. Beyreuther, R. E. Tanzi, C. L. Masters, and A. I. Bush, personal communication. Hence, an elaboration of the interactions between Aβ and zinc in vitro may be germane to the pathology of Alzheimer's disease. The concentration of zinc required to precipitate Aβ1–40 in vitro has been in disagreement with results reported recently by Esler et al. (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar), who have claimed that concentrations no less than 100 μm are required to demonstrate appreciable precipitation of the peptide. In particular, the validity of the filtration assay for Aβ aggregation used in our previous study was challenged by these workers who contended that125I-Aβ1–40 is a suitable tracer for monitoring the interaction of Aβ with zinc, at variance with our findings (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar). Rodriguez et al. (27.J. Biol. Chem. 272, 21037–21044Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S., and Glabe, C. G. J. Biol. Chem., 272, 21037–21044.Google Scholar) have recently reported that zinc concentrations below 100 μm induce the abundant and immediate precipitation of soluble Aβ1–40, in agreement with our initial reports (16Bush A.I. Pettingell Jr., W.H. Paradis M. Tanzi R.E. J. Biol. Chem. 1994; 269: 12152-12158Abstract Full Text PDF PubMed Google Scholar, 19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar, 20Bush A.I. Moir R.D. Rosenkranz K.M. Tanzi R.E. Science. 1995; 268: 1921-1923Crossref PubMed Scopus (32) Google Scholar) and in disagreement with the findings of Esler et al. (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar). This debate is important for two reasons. First, if the concentration of zinc required to precipitate Aβ is, in fact, over 100 μm, then it is very unlikely that this interaction is of any neurobiological significance. Much of the zinc that is released by synaptic transmission (24Assaf S.Y. Chung S.-H. Nature. 1984; 308: 734-736Crossref PubMed Scopus (1046) Google Scholar, 25Howell G.A. Welch M.G. Frederickson C.J. Nature. 1984; 308: 736-738Crossref PubMed Scopus (713) Google Scholar) may not be available for exchange with Aβ since it will in large part be sequestered by macromolecules and other ligands (28Frederickson C.J. Int. Rev. Neurobiol. 1989; 31: 145-328Crossref PubMed Scopus (981) Google Scholar). If >100 μm zinc is required to induce Aβ precipitation, the majority of the zinc released during neurotransmission would need to exchange with Aβ if synaptic zinc were to be a factor in the peptide's accumulation. This is unlikely. If, on the other hand, low micromolar concentrations of zinc are required to precipitate Aβ, then only ≈1% of the total zinc released during glutamatergic neurotransmission would be required to induce Aβ assembly, making such an interaction far more likely. Second, our findings (19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar) questioned the use of 125I-Aβ as a valid tracer for unmodified Aβ behavior, and in our hands, the effect of low micromolar zinc upon the precipitation of Aβ differentiated the iodinated peptide from unmodified Aβ. This is important since radiolabeling of Aβ by iodination is a common means of creating a marker for the peptide. To explore the reasons for the variance between our findings and those of Esler et al. (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar), we studied physicochemical factors that influence the interaction of zinc with synthetic Aβ1–40, and used turbidometry as a highly specific, although relatively insensitive, method for monitoring peptide aggregation. We now report that the striking precipitation of Aβ1–40 by low micromolar concentrations of zinc is sensitive to complex factors in the buffer milieu that impact upon the peptide's conformation and polymerization state. These factors may explain the variance that exist between our earlier findings and the report of Esler et al. (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar). Our findings also indicate that the conformational state of the peptide may not be perturbed by precipitation with zinc. These data may provide insights into neurobiological factors that influence the solubility of Aβ in the pathophysiological environment of the brain in Alzheimer's disease. Human Aβ1–40 amyloid peptide was used in all experiments, synthesized, purified, and characterized by HPLC 2The abbreviations used are: HPLC, high performance liquid chromatography; TBS, Tris-buffered saline; MES, 4-morpholineethanesulfonic acid; TFE, trifluoroethanol; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; ZBD, zinc binding domain; MOPS, 4-morpholinepropanesulfonic acid; APP, amyloid protein precursor. analysis, amino acid analysis and mass spectroscopy by W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT). The HPLC elution profile of Aβ1–40 peptide in preparations used in these experiments was identified as a sole peak in the eluate. Amino acid analysis of the synthetic peptide indicated that there were no apparent chemical modifications at amino acid residues. Mass spectroscopy was performed on each batch of peptide as a further confirmation. Milli-Q water (Millipore Corp., Milford, MA) was used for Aβ solubilization and other stock reagent dilution. Buffers were treated with Chelex-100 resin (Bio-Rad) to minimize trace metal contamination, and filtered through a 0.22-μm cellulose acetate filter unit (Corning Costar Corporation, Cambridge, MA). The background zinc concentrations in the buffers were measured at <0.1 μm by ion-coupled plasma-atomic emission spectroscopy. Standard zinc stock solution (10 mg/ml in 10% HCl, U. S. National Institute of Standards and Technology, Gaithersburg, MD) was used in all experiments. All other reagents were at least analytical grade. Synthetic Aβ peptide solutions were prepared on the day of the experiment according to the protocol of Evans et al. (29Evans K.C. Berger E.P. Cho C.-G. Weisgraber K.H. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 763-767Crossref PubMed Scopus (345) Google Scholar), except where indicated. Lyophilized peptide was first solubilized in water to reach 500 μm and then indirectly sonicated for 3 min (30 s on, 10 s off) through a water bath to avoid frothing. The peptide preparation was then filtered through a water-washed Spin-X cellulose acetate filter unit (0.22 μ, Corning Costar Corporation). Sonication and filtration are considered to be critical procedures to remove any trace of peptide microparticulate matter. Concentrations of Aβ1–40 were determined by BCA protein assay (Pierce). The validity of the BCA assay for the measurement of Aβ peptide concentrations in these solutions was confirmed by amino acid analysis. Experiments were performed using Waters model 650E system (Millipore Corporation, Milford, MA) connected to a column (Bio-Rad Econo-Column, 30 × 1.0 cm) prepacked with Superdex 30 (Pharmacia Biotech AB, Uppsala, Sweden). Aβ1–40 (0.5 ml, 2.3 μm) in Tris-HCl-buffered saline (TBS, 20 mm Tris, 150 mm NaCl, pH 7.4) was injected into the column preequilibrated with TBS at room temperature and eluted at 0.5 ml/min. The column was calibrated with combined gel-filtration molecular mass markers (Bio-Rad and Sigma), vitamin B12 (1.35 kDa), aprotinin (6.5 kDa), cytochrome C (12.4 kDa), equine myoglobin (17 kDa), and carbonic anhydrase (29 kDa). The total volume and void volume of the column were determined by elution volumes of dichromate anion (0.22 kDa) and blue dextran (2000 kDa), respectively. The Aβ1–40 elution peak was monitored at 214-nm absorbance, and the amount of Aβ1–40 eluting from the column was estimated by calibrating the absorbance of Aβ at 214 nm against known peptide concentrations. Turbidity measurement as an assay for aggregation was performed according to established protocols (29Evans K.C. Berger E.P. Cho C.-G. Weisgraber K.H. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 763-767Crossref PubMed Scopus (345) Google Scholar, 30Jarrett J.T. Lansbury Jr., P.T. Biochemistry. 1992; 31: 12345-12352Crossref PubMed Scopus (281) Google Scholar, 31Come J.H. Fraser P.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5959-5963Crossref PubMed Scopus (362) Google Scholar, 32Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1790) Google Scholar) with minor modifications. The reactions were performed at room temperature in a flat-bottom 96-well microtiter plate (Corning Costar Corporation), and absorbances (405 nm) were measured using a V maxkinetic microplate reader directed by Softmax version 2.32 software (Molecular Devices Corporation). Automatic 30-s plate agitation mode was selected for the plate reader to evenly suspend the aggregates in the wells before all readings. In most experiments, Aβ1–40 was brought to 10 μm (300 μl) in either 50 mm HEPES buffer, 150 mmNaCl, 0–300 μm zinc, pH 7.4, ±chelator, or 50 mm MES buffer (150 mm NaCl, pH 5.5), and incubated at 37 °C before absorbance measurements were taken at room temperature. To investigate the reversibility of zinc-induced Aβ1–40 aggregation, 25 μm zinc and 25 μm Aβ1–40 were mixed in 150 mm NaCl, 50 mm HEPES, pH 7.4 (200 μl), and turbidity measurements were taken at four 1-min intervals using a 96-well plate reader. Subsequently, 20-μl aliquots of 10 mm EDTA or 10 mm zinc (prepared in incubation buffer) were added into the wells alternately, and following a 2-min delay, a further four readings were taken at 1-min intervals. After the final EDTA addition and turbidity reading, the mixtures were incubated for an additional 30 min before taking final readings. To investigate the reversibility of pH 5.5-induced Aβ1–40 aggregation, Aβ1–40 was brought to 25 μm in 150 mm NaCl, 50 mm HEPES, pH 7.4 (200 μl), and its absorbance was read at 405 nm as the background reading. The pH of the solution was then brought to 5.5 by the addition of concentrated HCl (5.5 μl), and turbidity measurements were taken at four 1-min intervals. Subsequently, concentrated NaOH (7.5 μl) was added into the wells to adjust the pH back to 7.4, and following a 2-min delay, a further four measurements were taken at 1-min intervals. These cycles were repeated as indicated, and the pH of the mixture was constantly monitored with a pH probe. To further determine the state of aggregation of the incubated peptides in the reversibility experiments, replicate samples (300 μl) representing various zinc-containing or chelated conditions were removed from the incubation tray at various time points, pelleted (10,000 × g for 15 min), and either the supernatant was measured for remaining peptide content before and after centrifugation using the BCA assay, or the pellet was stained with 50 μl of Congo Red (1% in 50% ethanol for 5 min). Pellets were washed twice with 50% ethanol (100 μl) before being resuspended in 20 μl of HEPES buffer. An aliquot (3 μl) of each resuspension was placed on a microscope slide uniformly for microscopic analysis under polarized light. Stock solutions of Aβ1–40 (0.2 mm) were prepared by dissolving lyophilized peptide in either 20 mm Tris-HCl, pH 7.4, or 10–30% trifluoroethanol (TFE), or 75% dimethyl sulfoxide (Me2SO), 25% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (v/v), on the day of the study. Other stocks of Aβ1–40 dissolved in 75% Me2SO, 25% HFIP or water were stored at −20 °C for up to 2 months to determine the effects of storage upon zinc-induced aggregation. All peptide solutions were centrifuged (10,000 × g for 20 min) prior to use to remove aggregates. The Aβ1–40 stock solutions in Tris buffer and Me2SO/HFIP were brought to 2.3 μm with 150 mm NaCl, 20 mmTris-HCl, pH 7.4, ±zinc (0, 10, and 30 μm) and incubated (30 min, 37 °C). The Aβ1–40 stock solution dissolved in 10–30% TFE was brought to 2.3 μm with 150 mm NaCl, 20 mm Tris-HCl, 10–30% TFE, pH 7.4, ±zinc (0, 10, and 30 μm) and incubated (30 min, 37 °C). Following incubation, the mixtures were filtered through a 0.22-μm cellulose acetate filter, and the amount of peptide entering the filtrate was determined by micro BCA protein assay (Pierce), according to a modification of the Aβ aggregation assay developed in our group (19Bush A.I. Pettingell Jr., W.H. Multhaup G. Paradis M.D. Vonsattel J.-P. Gusella J.F. Beyreuther K. Masters C.L. Tanzi R.E. Science. 1994; 265: 1464-1467Crossref PubMed Scopus (1435) Google Scholar). To characterize synthetic Aβ1–40 in neutral buffered saline, gel-filtration chromatography was performed. Aβ1–40 solution (2.3 μm) was freshly prepared in TBS from its lyophilized powder (see "Experimental Procedures") on the day of the experiment. The peptide solution was loaded onto the gel-filtration column within 1 h of preparation, and eluted as a single sharp peak corresponding to 8.6 kDa compared to molecular size markers, and at an estimated concentration of 470 nm (Fig.1), compatible with the peptide being in a dimeric state. Other groups have reported the presence of dimeric Aβ using gel-filtration chromatography (33Hilbich C Kisters-Woike B. Reed J. Masters C.L. Beyreuther K. J. Mol. Biol. 1991; 218: 149-163Crossref PubMed Scopus (552) Google Scholar, 34Soreghan B. Kosmoski J. Glabe C. J. Biol. Chem. 1994; 269: 28551-28554Abstract Full Text PDF PubMed Google Scholar), and our data are in agreement with the recent report that Aβ1–40 is predominantly dimeric upon gel-filtration in neutral buffered saline (27.J. Biol. Chem. 272, 21037–21044Garzon-Rodriguez, W., Sepulveda-Becerra, M., Milton, S., and Glabe, C. G. J. Biol. Chem., 272, 21037–21044.Google Scholar). However, in the absence of NaCl, we found that the peptide's elution profile became too broad to allow its relative molecular size to be resolved (data not shown), suggesting that the apparent dimerization of the peptide is dependent upon the presence of NaCl. Since we wished to achieve a basic data set that describes the behavior of the most abundant species of Aβ under conditions that approach a physiologically plausible milieu, we proceeded to study the behavior of the peptide in isotonic neutral buffered saline, mindful that the concentration of NaCl appears to enact significant conformational effects upon the peptide. Some studies have reported chromatographic profiles of Aβ peptides that appear nondimeric (monomeric or oligomeric). Apart from differences in the composition of the solvent system used for the chromatographic procedure, these alternative results may have been due to differences in the behaviors of the specific subspecies of Aβ peptide that were studied, reported differences in the preparation of the Aβ peptide, and variations in the experimental procedure (35Barrow C.J. Yasuda A. Kenny P.T.M. Zagorski M.G. J. Mol. Biol. 1992; 225: 1075-1093Crossref PubMed Scopus (621) Google Scholar, 36Shen C.-L. Scott G.L. Merchant F. Murphy R.M. Biophys. J. 1993; 65: 2383-2395Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 37Shen C.-L. Fitzgerald M.C. Murphy R.M. Biophys. J. 1994; 67: 1238-1246Abstract Full Text PDF PubMed Scopus (76) Google Scholar). For example, in our previous study of Aβ1–40 by gel-filtration chromatography, we observed that the peptide migrated mainly as an apparent dimer (65%) together with minor apparent polymer (30%) and monomer (5%) peaks (16Bush A.I. Pettingell Jr., W.H. Paradis M. Tanzi R.E. J. Biol. Chem. 1994; 269: 12152-12158Abstract Full Text PDF PubMed Google Scholar). The difference between our current result and the previous result can be explained as due to two newly introduced variables. First, we have introduced sonication in the preparation of Aβ1–40 peptide solution, which may have contributed to the dissolution of Aβ1–40 polymer into dimeric or monomeric species. Second, in the current study only 5 μg of Aβ1–40 were applied to the column compared with 55 μg in the earlier study. Therefore, absorbance readings at 214 nm in this study are much closer to the base-line buffer absorbance reading, and the absorbance may not be sufficient to demonstrate a significant monomeric peak, since, based upon our earlier data, its proportion is expected to be small in the total Aβ peptide eluent. Therefore, in the current study the presence of a small proportion of monomeric peptide cannot be excluded. To confirm whether the concentration of zinc required to induce Aβ1–40 precipitation is in the low or high micromolar range, we studied the behavior of the peptide by turbidometry, because it is a well established method that has been used to study the aggregation state of Aβ (29Evans K.C. Berger E.P. Cho C.-G. Weisgraber K.H. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 763-767Crossref PubMed Scopus (345) Google Scholar, 30Jarrett J.T. Lansbury Jr., P.T. Biochemistry. 1992; 31: 12345-12352Crossref PubMed Scopus (281) Google Scholar, 31Come J.H. Fraser P.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5959-5963Crossref PubMed Scopus (362) Google Scholar, 32Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1790) Google Scholar). We first determined that the absorbance (405 nm) value of freshly prepared Aβ1–40 (10 μm) in TBS was equivalent to the absorbance of the experimental buffers used (data not shown), indicating that the presence of the soluble Aβ peptide does not contribute to turbidity in this system at the time frame studied. We chose the minimal zinc binding domain (ZBD) at the amino terminus of APP, which has a K a for zinc binding of ≈750 nm (ZBD, residues 179–189 of APP) (38Bush A.I. Multhaup G. Moir R.D. Williamson T.G. Small D.H. Rumble B. Pollwein P. Beyreuther K. Masters C.L. J. Biol. Chem. 1993; 268: 16109-16112Abstract Full Text PDF PubMed Google Scholar), as a zinc-binding control peptide for comparison to the behavior of Aβ1–40 in the experiments. The background absorbance turbidity of ZBD (10 μm in TBS, pH 7.4) was found to be of the same as that of Aβ1–40 in TBS alone (data not shown), and incubation of ZBD solutions with the zinc concentrations used in this study did not increase their turbidity, indicating that the ZBD peptide does not aggregate in the presence of zinc. We proceeded to study conditions representing concentrations of peptide (10 μm) and zinc (<100 μm) for which Esleret al. (26Esler W.P. Stimson E.R. Jennings J.M. Ghilardi J.R. Mantyh P.W. Maggio J.E. J. Neurochem. 1996; 66: 723-732Crossref PubMed Scopus (126) Google Scholar) could find no evidence of aggregation. There were negligible changes of absorbance readings for solution of 10 μm Aβ1–40 mixed with 0.1, 0.5 and 1 μmzinc, compared with 10 μm Aβ1–40 alone. However, the absorbance increases of Aβ1–40 with 5 and 10 μm zinc were substantially above background (Fig.2 A), corroborat
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