ISOLATION OF A PHOSPHORYLATED SOLUBLE TAU FRACTION FROM ALZHEIMERS-DISEASE BRAIN

Modified forms of tau proteins are major components of the paired heli cal filaments (PHFs) present in Alzheimer brains. In this study, tau f rom cytosolic samples obtained from normal and Alzheimer disease brain s were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents usi ng an stepwise pH gradient. Immunoblot analysis of the different fract ions using antibody Tau-1 (recognizing an unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recog nizing a phosphorylated epitope in PHF-tau) have been carried out. Pho sphorylated tau species (Tan 1-nonreactive and SMI 31-reactive) are on ly isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylatio n by proline-directed protein kinases are not further phosphorylated i n vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, sol uble 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.