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• W2141904030 abstract "Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n= 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32–41 and 76–77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72–77 and 65–82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929–30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD. Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n= 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32–41 and 76–77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72–77 and 65–82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929–30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD. The pathological hallmark of Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; APP, amyloid protein precursor; APP-KPI+, amyloid protein precursor isoforms containing a Kunitz protease inhibitory domain; EGFBP, epidermal growth factor-binding protein; KPI, Kunitz protease inhibitory domain; mAb, monoclonal antibody; pAb, polyclonal antibody; LRP, low density lipoprotein receptor-related protein; sAPP, soluble COOH-terminal truncated amyloid protein precursor; PAGE, polyacrylamide gel electrophoresis. is the deposition of amyloid as cerebrovascular, diffuse and neuritic plaques (within the brain extracellular space), and neurofibrillary tangles (within neurons). The principal component of extracellular amyloid is a 4-kDa peptide, the Aβ protein (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Google 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: 4245-4249Google Scholar) (also called the βA4 protein). The Aβ peptide is not expressed as a functional protein entity (3Citron M. Haass C. Selkoe D.J. Neurobiol. Aging. 1993; 14: 571-573Google Scholar) but is released by the processing of a much larger transmembrane protein, the amyloid protein precursor (APP). The pathogenesis of AD is thought to involve the disregulated expression or abnormal processing of APP. APP is encoded by a single 18-exon gene on chromosome 21 (4Kang J. Lemaire H. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K. Multhaup G. Beyreuther K. Müller-Hill B. Nature. 1987; 325: 733-736Google Scholar, 5Goldgaber D. Lerman M.I. McBride O.W. Saffiotti U. Gajdusek D.C. Science. 1987; 235: 877-880Google Scholar, 6Tanzi R.E. Gusella J.F. Watkins P.C. Bruns G.A.P. St George-Hyslop P. van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Google Scholar). Exons 7, 8, and 15 of the APP gene can be alternatively spliced to produce multiple isoforms. In brain the predominant isoform transcripts demonstrated to date are APP695, APP751, and APP770 (7Kitaguchi N. Takahashi Y. Tokushima Y. Shiojiri S. Ito H. Nature. 1988; 331: 530-532Google Scholar, 8Tanzi R.E. McClatchey A.I. Lamperti E.D. Villa-Komaroff L. Gusella J.F. Neve R.L. Nature. 1988; 331: 528-530Scopus (872) Google Scholar, 9Ponte P. Gonzalez-DeWhitt P. Schilling J. Miller J. Hsu D. Greenberg B. Davis K. Wallace W. Lieberburg I. Fuller F. Cordell B. Nature. 1988; 331: 525-527Google Scholar). These transcripts code for species containing 695, 751, and 770 amino acids, respectively. The isoforms APP751 and APP770 both contain a Kunitz protease inhibitory (KPI) motif that APP695 lacks. APP770 contains an additional OX.2 domain (7Kitaguchi N. Takahashi Y. Tokushima Y. Shiojiri S. Ito H. Nature. 1988; 331: 530-532Google Scholar, 10Weidemann A. König G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Google Scholar). The secreted form of APP751 is identical to protease nexin II, a plasma serine protease inhibitor (11Oltersdorf T. Fritz L.C. Schenk D.B. Lieberburg I. Johnson-Wood K.L. Beattie E.C. Ward P.J. Blacher R.W. Dovey H.F. Sinha S. Nature. 1989; 341: 144-147Google Scholar). In addition to KPI and OX.2 domains several other structural features have been identified on APP, including binding domains for heparin (12Small D.H. Nurcombe V. Reed G. Clarris H. Moir R. Beyreuther K. Masters C.L. J. Neurosci. 1994; 14: 2117-2127Google Scholar), zinc (13Bush 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: 16109-16112Google Scholar, 14Bush A.I. Pettingell W.H. de Paradis M. Tanzi R.E. Wasco W. J. Biol. Chem. 1994; 269: 26618-26621Google Scholar) and copper (15Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Google Scholar), and N-linked carbohydrate attachment sites (10Weidemann A. König G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Google Scholar). Normal catabolism of APP involves proteolytic cleavage of full-length membrane-associated forms within the extracellular domain of the Aβ region and release of soluble COOH-terminal truncated species (sAPP) (16Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Google Scholar, 17Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Google Scholar). The proteases that release sAPP have yet to be identified but have been named the α- and β-secretases. The α-secretase cleaves within the Aβ sequence of APP and its products are non-amyloidogenic. The β-secretase cleavage site is the NH2 terminus of the Aβ domain. The proteolytic activities that release intact Aβ from the transmembrane domain of APP (COOH terminus of Aβ) have been designated γ-secretases. The catabolic pathway for Aβ generation is unclear but probably involves internalization of full-length APP from the cell surface and degradation in endosomal-lysosomal complexes (18Estus S. Golde T.E. Kunishita T. Blades D. Lowery D. Eisen M. Usiak M. Qu X. Tabira T. Greenberg B.D. Younkin S.G. Science. 1992; 255: 726-728Google Scholar, 19Golde T.E. Estus S. Younkin L.H. Selkoe D.J. Younkin S.G. Science. 1992; 255: 728-730Google Scholar, 20Haass C. Koo E.H. Mellon A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Google Scholar). In cultured hamster cells one route for Aβ production involves a coated pit-mediated endocytic pathway (21Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Google Scholar). Low density lipoprotein receptor-related protein (LRP) has recently been shown to also mediate the internalization and degradation of KPI-containing (KPI+) APP forms (22Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Google Scholar, 23Knauer M.F. Orlando R.A. Glabe C.G. Brain Res. 1996; 740: 6-14Google Scholar). Internalization via LRP has been shown to increase if APP is complexed to the binding protein for epidermal growth factor (EGFBP) (23Knauer M.F. Orlando R.A. Glabe C.G. Brain Res. 1996; 740: 6-14Google Scholar, 24Van Nostrand W.E. Cunningham D.D. J. Biol. Chem. 1987; 262: 8508-8514Google Scholar). EGFBP is specific for APP forms that contain a KPI domain and does not form complexes with APP695. At present it is unclear if LRP mediated internalization of APP leads to Aβ production. Transcripts for APP695 are expressed almost exclusively in brain where they are a major neuronal APP mRNA species (25Neve R.L. Boyce F.M. McPhie D.L. Greenan J. Oster-Granite M.L. Neurobiol. Aging. 1996; 17: 191-203Google Scholar, 26Golde T.E. Estus S. Usiak M. Younkin L.H. Younkin S.G. Neuron. 1990; 4: 253-267Google Scholar). The KPI+ isoforms APP751 and APP770are expressed in both the central nervous system and in peripheral tissues. Several lines of evidence suggest that expression levels of KPI+ isoforms of APP may be important in AD pathogenesis. There is evidence that the cerebral APP751/APP695 mRNA ratio increases with age (27Tanaka S. Liu L. Kimura J. Shiojiri S. Takahashi Y. Kitaguchi N. Nakamura S. Ueda K. Mol. Brain Res. 1992; 15: 303-310Google Scholar, 28Tanaka S. Nakamura S. Kimura L. Ueda K. Neurosci. Lett. 1993; 163: 19-21Google Scholar). Although reports are conflicting, mRNA levels for KPI+ APP751 and APP770 species may be elevated in the brains (6Tanzi R.E. Gusella J.F. Watkins P.C. Bruns G.A.P. St George-Hyslop P. van Keuren M.L. Patterson D. Pagan S. Kurnit D.M. Neve R.L. Science. 1987; 235: 880-884Google Scholar, 8Tanzi R.E. McClatchey A.I. Lamperti E.D. Villa-Komaroff L. Gusella J.F. Neve R.L. Nature. 1988; 331: 528-530Scopus (872) Google Scholar, 29Johnson S.A. Pasinetti G.M. May P.C. Ponte P.A. Cordell B. Finch C.E. Exp. Neurol. 1988; 102: 264-268Google Scholar, 30Johnson S.A. Rogers J. Finch C.E. Neurobiol. Aging. 1989; 10: 267-272Google Scholar, 31Spillantini M.G. Hunt S.P. Ulrich J. Goedert M. Mol. Brain Res. 1989; 6: 143-150Google Scholar, 32Johnson S.A. McNeill T. Cordell B. Finch C.E. Science. 1990; 248: 854-857Google Scholar, 33Neve R.L. Finch E.A. Dawes L.R. Neuron. 1988; 1: 669-677Google Scholar, 34Tanaka S. Nakamura S. Ueda K. Kameyama M. Shiojiri S. Takahashi Y. Kitaguchi N. Ito H. Biochem. Biophys. Res. Commun. 1988; 157: 472-479Google Scholar, 35Tanaka S. Shiojiri S. Takahashi Y. Kitaguchi N. Ito H. Kameyama M. Kimura J. Nakamura S. Ueda K. Biochem. Biophys. Res. Commun. 1989; 165: 1406-1414Google Scholar, 36Johnson S.A. Norgren S. Ravid R. Wasco W. Winblad B. Lannfelt L. Cowburn R.F. Mol. Brain Res. 1996; 43: 85-95Google Scholar) and fibroblasts (37Okada A. Urakami K. Takahashi K. Ohno K. Sato K. Endo H. Dementia. 1994; 5: 55-56Google Scholar, 38Rockenstein E.M. McConlogue L. Tan H. Power M. Masliah E. Mucke L. J. Biol. Chem. 1995; 270: 28257-28267Google Scholar) of AD patients. The cerebrospinal fluid of AD patients may also have elevated levels of KPI+ forms of sAPP protein (39Kitaguchi N. Tokushima Y. Oishi K. Takahashi Y. Shiojiri S. Nakamura S. Tanaka S. Kodaira R. Ito H. Biochem. Biophys. Res. Commun. 1990; 166: 1453-1459Google Scholar, 40Palmert M.R. Usiak M. Mayeux R. Raskind M. Tourtellotte W.W. Younkin S.G. Neurology. 1990; 40: 1028-1034Google Scholar). APP-KPI+ immunoreactivity has be reported to correlate with neuritic plaque density (41Zhan S.S. Sandbrink R. Beyreuther K. Schmitt H.P. Clin. Neuropathol. 1995; 14: 142-149Google Scholar). In studies using animal models, brain transcripts for APP-KPI+ isoforms were found to increase in response to trauma (42Abe K. St. George-Hyslop P.H. Tanzi R.E. Kogure K. Neurosci. Lett. 1991; 125: 172-174Google Scholar, 43Kalaria R.N. Bhatti S.U. Palatinsky E.A. Pennington D.H. Shelton E.R. Chan H.W. Perry G. Lust W.D. Neuroreport. 1993; 4: 211-214Google Scholar, 44Solá C. Garcı́a-Landon F.J. Mengod G. Probst A. Frey P. Palacios J.M. Mol. Brain Res. 1993; 17: 41-52Google Scholar, 45Willoughby D.A. Johnson S.A. Pasinetti G.M. Tocco G. Najm I. Baudry M. Finch C.E. Exp. Neurol. 1992; 118: 332-339Google Scholar) and transgenic mice over-expressing KPI+ species of APP show age-related learning deficits (46Morgan P.M. Higgins L.S. Cordell B. Moser P.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5341-5345Google Scholar). A recently developed transgenic mouse that exhibits extensive cerebral Aβ amyloid deposition (47Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Guido T. Hagopian S. Johnson-Wood K. Khan K. Lee M. Leibowitz P. Lieberburg I. Little S. Masliah E. McConlogue L. Montoya-Zavala M. Mucke L. Paganini L. Penniman E. Power M. Schenk D. Seubert P. Snyder B. Soriano F. Tan H. Vitale J. Wadsworth S. Wolozin B. Zhao J. Nature. 1995; 373: 523-527Scopus (2247) Google Scholar) has been reported to overexpress human APP-KPI+ mRNA and have suppressed endogenous APP-KPI− message levels (38Rockenstein E.M. McConlogue L. Tan H. Power M. Masliah E. Mucke L. J. Biol. Chem. 1995; 270: 28257-28267Google Scholar). Finally, it has recently been confirmed that KPI+ isoforms of APP are more amyloidogenic, at least in transfected cells (48Ho L. Fukuchi K. Younkin S.G. J. Biol. Chem. 1996; 271: 30929-30934Google Scholar). At present it is unclear if an increase in KPI+ isoforms of APP occurs at the protein level in the brains of AD patients. In this study, APP was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of AD and normal brains and then compared for KPI+ isoforms by two different assay methods. We find that the level of APP-KPI+ in purified material from the soluble subcellular fraction of AD brains was significantly elevated (doubled) relative to control patients. Lyophilized purified trypsin from bovine pancreas and carbobenzoxy-valyl-glycyl-arginine-4-nitranilide acetate (Chromozym TRY) were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Electrophoretic molecular weight markers were purchased from Amersham (Buckinghamshire, United Kingdom). Electrophoresis reagents were from Bio-Rad and polyvinylidene difluoride membrane from Millipore Corp. (Bedford, MA). Bicinchoninic acid (BCA) protein assay reagent was from Pierce (Rockford, IL). Protein A-Sepharose and other chromatographic resins were from Pharmacia (Uppsala, Sweden). The monoclonal antibody (mAb) 22C11 (available from Boehringer Mannheim GmbH) and polyclonal (pAb) R45 rabbit antiserum recognize an epitope near the amino terminus of APP (10Weidemann A. König G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Google Scholar, 49Hilbich C. Mönning U. Grund C. Masters C.L. Beyreuther K. J. Biol. Chem. 1993; 268: 26571-26577Google Scholar). MAb 7H5 was raised against a bacterial construct encompassing the KPI domain (residues 289–347 of APP770) and was kindly provided by Dr. Dale Schenk, Athena Neurosciences. Secondary antibodies used in Western blots were purified IgG fractions of anti-mouse antibodies conjugated to alkaline phosphatase (Promega, Sydney, Australia). Human brains were obtained 12–24 h post mortem. At the time of autopsy one cerebral hemisphere was frozen at −70 °C and the other hemisphere was fixed in formalin for histological examination. The clinical diagnosis of Alzheimer's disease was confirmed by subsequent histological evidence of amyloid plaques or neurofibrillary tangles. Neurological control patients were clinically diagnosed as having Parkinson's disease or Huntington's disease with subsequent pathological confirmation. All clinical groups were age matched. APP was purified to homogeneity from the ultracentrifuge soluble fraction and Triton X-100 membrane pellet extract of human brain homogenate according to the method of Moir et al. (50Moir R.D. Martins R.N. Small D.H. Bush A.I. Milward E.A. Multhaup G. Beyreuther K. Masters C.L. J. Neurochem. 1992; 59: 1490-1498Google Scholar). Briefly, isolated human brain cortex was homogenized and centrifuged. The supernatant (soluble subcellular fraction) was removed and the pellet incubated with Triton X-100. Insoluble material was pelleted by a second centrifugation and the supernatant (membrane extract) removed. Purification of APP from the crude brain subcellular fractions involves a sequential series of chromatographic steps: anion exchange (MacroPrep high Q resin), affinity chromatography (heparin-Sepharose gel), a second anion exchange step (Mono Q column) and hydrophobic interaction chromatography (phenyl-Superose column). Peak APP fractions from the final phenyl-Superose elute were concentrated and desalted into 20 mm Tris, pH 7.4, and 150 mm NaCl (TBS) on Centricon 30 Protein Concentrators (Amicon, Beverly, MA). APP was monitored at each purification step by mAb 22C11 immunoblots. Total APP protein yields were determined by amino acid analysis and BCA assay. MacroPrep high Q column effluent was titrated against corresponding starting material (crude brain subcellular fractions) on mAb 22C11 Western blots. Crude starting material and MacroPrep high Q column effluent is unsuited to analysis by direct immunoblotting on our Western blotting system because of severe band distortion and poor resolution on SDS-PAGE caused by the relatively large sample volumes and high protein loading required for a detectable APP signal. Therefore, all samples were concentrated by immunoprecipitation with pAb R45 (raised against the same epitope as mAb 22C11) before electrophoresis. PAb R45 immunoprecipitates prepared from increasing volumes of MacroPrep high Q effluent (500–1500 μl in 100-μl increments) and 75 μl of corresponding starting material were compared on mAb 22C11 Western blots by reflectance analysis. Purification efficiency was calculated from the volume of column effluent observed to give an equivalent signal (sum of all bands) to the sample of starting material. Calculations included an adjustment for the change in sample volume during chromatography. Samples were also analyzed to determine if 100–110- or 120–130-kDa immunoreactive species are selectively absorbed by the MacroPrep high resin. For this analysis, effluent concentrations with similar total APP signals as starting material were selected. The density of the 100–110- and 120–130-kDa bands were determined for the selected lanes. The ratio of the upper band density to lower band density (density of the upper band/density of the lower band) was then calculated for starting material and corresponding column effluents. MacroPrep high Q and desalted phenyl-Superose eluates were also compared on mAb 22C11 immunoblots. Since APP signal in chromatographic eluates is readily detected without prior concentration, these samples were not immunoprecipitated with pAb R45 before electrophoresis. The ratio of 100–110- to 120–130-kDa immunoreactive material in the eluates was calculated as described previously for immunoprecipitates of crude brain subcellular fractions and MacroPrep high Q effluents. Trypsin (30 ng) was incubated with or without APP in TBS (175 μl) for 20 min at 20 °C in 96-well microtiter plates. Trypsin activity was then determined in each well by the addition of 25 μl of the synthetic trypsin substrate Chromozym TRY (4 mm) and monitoring the change in absorbance (415 nm) over 20 min. Under the conditions used, assay linearity was maintained for at least 40 min after the addition of substrate and no increase in absorbance was detected in the presence of APP alone. Samples for immunoprecipitation were incubated overnight at 4 °C with pAb R45 or mAb 7H5 in TBS containing 1% bovine serum albumin and 0.1% Tween 20. R45·antigen complexes were precipitated by addition of protein A-Sepharose (10 μl of swollen beads). For precipitation of 7H5·antigen complexes, protein A-Sepharose was first preincubated with rabbit anti-mouse IgG to increase capture efficiency. The Sepharose beads were washed three times prior to release of immunoprecipitated material from the resin by addition of 100 μl of SDS-PAGE sample buffer. Samples were electrophoresed on SDS-PAGE (10% acrylamide gels) and blotted to polyvinylidene difluoride membrane. Membranes were blocked with bovine serum albumin (3%) before incubation with mAb 22C11 (1:3000) or mAb 7H5 (1:1000) for 3 h at room temperature. Following a 2-h incubation at room temperature with anti-mouse IgG coupled to alkaline phosphatase (1:10000), blots were developed for 15 min with Fast Red (3 mg/ml) and naphthol (0.2 mg/ml) in 100 mm NaCl, 5 mm MgCl2, and 100 mm Tris-HCl at pH 8.0. Quantitation of APP on protein blots were determined by reflectance analysis as described by Bush et al. (51Bush A.I. Whyte S. Thomas L.D. Williamson T.G. Van Tiggelen C.J. Currie J. Small D.H. Moir R.D. Li Q.X. Rumble B. Mönning U. Beyreuther K. Masters C.L Ann. Neurol. 1992; 32: 57-65Google Scholar). APP was purified to homogeneity from the soluble subcellular fraction and Triton X-100 membrane pellet extract of cerebral cortex isolated from AD (n = 10), normal (n = 7), and neurological control (three Huntington's disease and two Parkinson's disease cases) brains. Purity was confirmed by protein staining of SDS-PAGE gels using silver and by NH2-terminal sequencing. Purified APP samples contained one minor (80–90 kDa) and two major (100–110 and 120–130 kDa) bands on mAb 22C11 Western blots and silver-stained SDS-PAGE gels. Identity of each band was confirmed by amino-terminal sequencing. Starting material, chromatographic fractions, and purified protein from AD and control brains were compared and monitored for selective loss or concentration of APP species during purification. APP-KPI+levels in the test brains used in these experiments are typical for their respective clinical groups. Initial experiments compared crude brains subcellular fractions and corresponding column effluents from the first chromatographic step (MacroPrep high Q column). Under the conditions used, positive absorption of mAb 22C11 immunoreactive material by MacroPrep high Q resin is greater than 90% for both soluble and detergent extract fractions of control and AD brains (Table I). Addition of eluates back to the effluents restored APP signal to that of corresponding starting material (data not shown). Consistent with uniform absorption of APP forms by the MacroPrep high Q resin, immunoprecipitates of starting material and chromatographic effluents contain similar relative amounts of 100–110- and 120–130-kDa species (Table II). Eluates from the MacroPrep high Q column and the last purification step also have similar relative amounts of the two major APP bands (Table II). In addition, Western blot analysis at each chromatographic step found no evidence of proteolytic degradation of APP during purification. These results are concordant with close conservation of the relative amounts of different APP forms during purification of both control and AD subcellular fractions.Table IAPP absorption from AD and control brain subcellular fractions by MacroPrep high Q anion exchange resinSoluble fractionMembrane fractionControlADControlAD>92%1-a% of total APP captured from brain subcellular fractions by MacroPrep high Q column (first chromatographic step).>92%>91%>90%1-a % of total APP captured from brain subcellular fractions by MacroPrep high Q column (first chromatographic step). Open table in a new tab Table IIRecovery of purified APP from AD and control brainChromatographic sampleRatio of major bands (upper band/lower band)Control brainAD brainSolubleMembraneSolubleMembraneImmunoprecipitated ppt of starting material0.46 ± 0.090.54 ± 0.080.72 ± 0.130.66 ± 0.15Immunoprecipitated ppt of eluate from first step0.49 ± 0.070.52 ± 0.130.65 ± 0.100.56 ± 0.09Eluate from MacroPrep high Q (first step)0.40 ± 0.060.51 ± 0.100.69 ± 0.120.55 ± 0.08Eluate from phenyl-Superose (last step)0.44 ± 0.040.54 ± 0.110.63 ± 0.140.52 ± 0.02 Open table in a new tab Data showing the average yield of cortex and purified APP protein for each clinical group is summarized in Table III. AD brains yield significantly less cortex (p = 0.0011 by t test) per hemisphere than pooled controls. The yield of purified APP protein is also significantly less for AD brains (p = 0.0023). Table III also shows the yield of APP for each clinical group after normalizing for the reduced mass of cortex recovered from AD brains (APP yield per g of cortex). Recovery of purified APP from AD brains remains significantly less than controls after normalizing for the different yields in cortex, although the difference is barely significant at the p = 0.05 level (p = 0.048). The subcellular distribution of APP is similar for all clinical groups, with equivalent soluble and membrane extract fractions yielding similar amounts of purified material. These results are consistent with the widespread neuronal loss associated with AD.Table IIIYields of cerebral cortex and purified APP protein from control and AD brainsnYield of cortex per hemisphereAPP yield soluble fractionAPP yield membrane fractionTotal APP yield per hemispheregμg/g of cortexμgNormal controls7370 ± 800.99 ± 0.210.98 ± 0.29744 ± 258Neurological controls5390 ± 821.04 ± 0.251.10 ± 0.17825 ± 142Pooled controls12377 ± 813-aPooled control cortex versus AD cortex;p = 0.0011.1.01 ± 0.231.02 ± 0.26773 ± 2263-bPooled control total APP versus AD total APP;p = 0.0023.AD10270 ± 383-aPooled control cortex versus AD cortex;p = 0.0011.0.83 ± 0.260.89 ± 0.30463 ± 1513-bPooled control total APP versus AD total APP;p = 0.0023.3-a Pooled control cortex versus AD cortex;p = 0.0011.3-b Pooled control total APP versus AD total APP;p = 0.0023. Open table in a new tab Two methods were used to determine the amount of KPI+isoforms in the purified APP samples. The first assay exploits the inhibitory action of KPI+ isoforms on trypsin. The second KPI+ isoform assay employs immunoblotting techniques and anti-NH2-terminal (mAb 22C11) and -KPI domain (mAb 7H5) antibodies. Trypsin was preincubated for 20 min with purified APP and then assayed for activity using the synthetic trypsin substrate Chromozym TRY. Initial experiments confirmed that trypsin activity decreased linearly with increasing concentrations of both soluble and membrane-associated APP (Fig. 1). The purified APP samples from normal, AD, and neurological disease control brains were then compared for trypsin inhibition. The proportion of KPI+ isoforms in each sample was calculated from the inhibition of trypsin (30 ng) after preincubation with 50 ng of purified protein (Table IV). The calculations assume that trypsin and KPI+ forms of APP interact with a 1:1 stoichiometry (52Kitaguchi N. Takahashi Y. Oishi K. Shiojiri S. Tokushima Y. Utsunomiya T. Ito H. Biochim. Biophys. Acta. 1990; 1038: 105-113Google Scholar, 53Hynes T.R. Randal M. Kennedy L.A. Eigenbrot C. Kossiakoff A.A. Biochemistry. 1990; 29: 10018-10022Google Scholar), that total binding approaches 100% and that the average molecular mass of APP is 80 kDa and trypsin 24 kDa (based on the amino acid sequences of these proteins). Soluble and membrane-associated APP purified from control brain (pooled) have significantly different levels (p < 0.0001) of KPI+ isoforms. Membrane-associated material is predominantly KPI+, while most APP purified from the soluble fraction is KPI−. Both normal and neurological disease control groups display similar subcellular distributions of KPI+ isoforms. However, for AD brains, soluble and membrane-associated APP contain similar levels of KPI+ material. Furthermore, KPI+ isoforms in APP from the soluble subcellular fraction of AD brains are significantly elevated (p = 0.0014) compared with corresponding material from pooled controls. The level of KPI+ isoforms in membrane-associated APP from AD-affected patients is not significantly different from control subjects.Table IVTrypsin inhibition by purified APP from AD and control brainsSubcellular fraction% KPI+ APP; trypsin inhibitionNormal control (n = 7)Diseased control (n = 5)Pooled control (n = 12)AD (n = 10)Soluble31 ± 734 ± 2132 ± 154-aPooled control soluble fraction versuspooled control membrane fraction; p < 0.0001.,4-bPooled control soluble fraction versus AD-soluble fraction; p = 0.0014.76 ± 294-bPooled control soluble fraction versus AD-soluble fraction; p = 0.0014.Membrane81 ± 1770 ± 1777 ± 184-aPooled control soluble fraction versuspooled control membrane fraction; p < 0.0001.82 ± 204-a Pooled control soluble fraction versuspooled control membrane fraction; p < 0.0001.4-b Pooled control soluble fraction versus AD-soluble fraction; p = 0.0014. Open table in a new tab" @default.
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• W2141904030 title "Relative Increase in Alzheimer's Disease of Soluble Forms of Cerebral Aβ Amyloid Protein Precursor Containing the Kunitz Protease Inhibitory Domain" @default.
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