Isolation of a phosphorylated soluble tau fraction from Alzheimer's disease brain.
Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing an unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autùnoma de Madrid, Spain.
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MD
Ledesma
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Avila
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Correas
D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001923:Brain Chemistry; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003600:Cytosol / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D010766:Phosphorylation; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012995:Solubility; D016875:tau Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D016875:tau Proteins; C085348:tau-1 monoclonal antibody; D038461:Proline-Directed Protein Kinases; D017346:Protein Serine-Threonine Kinases