Abstract
The major protein subunit of the paired helical filaments (PHF) of Alzheimer disease (AD) is the microtubule-associated protein tau. Tau is a family of phosphopolypeptides that are abnormally phosphorylated in PHF. In this study, a non-PHF pool of tau abnormally phosphorylated at Ser-199/202, and tau not phosphorylated at this site (AD P-tau and AD tau, respectively) were isolated from the 27,000 x g to 200,000 x g fraction of AD brain homogenate by extraction in 8 M urea, followed by dialysis against Tris buffer. AD P-tau and AD tau were further purified and separated from each other by acid precipitation, glial fibrillary acidic protein affinity chromatography, and phosphocellulose chromatography. The resulting AD P-tau and AD tau preparations were free of cytoskeletal proteins, ubiquitin, and beta-amyloid peptide. Immunochemical and morphological analysis of AD P-tau preparations revealed that most of the protein was of non-PHF origin. The AD P-tau was about 3-4-fold (approximately 8 mol P04/mol protein, M(r) 41,318) more phosphorylated than cytosolic tau from AD and control brains. Unlike PHF, the AD P-tau lacked ubiquitin. In AD brain the levels of cytosolic tau were about half of those in control aged cases. These findings suggest that the abnormal phosphorylation of tau in AD occurs in the cytosol.
Highlights
The major protein subunit of the paired helical filaments (PHF) of Alzheimer disease (AD) is the microtubule-associated protein tau
A non-PHF pool of tau abnormally phosphorylated at Ser-199/202, and tau not phosphorylated at this site (AD P-tau and AD tau, respectively)were isolated from the 27,000 X g to 200,000 X g fraction of AD brain homogenate by extraction in 8 M urea, followed by dialysis against Tris buffer
The SedimentableNon-PHF Taufrom AD Brain Contains BothAbnormallyPhosphorylatedand ApparentlyNormal Species-There are four pools of tau in AD brain, the PHF, the AD P-tau, the AD tau, and the c-tau. Both PHF and AD Ptau are abnormally phosphorylated at Ser-199/202 so that they require dephosphorylation to react with Tau-1 antibody; the AD P-tau is soluble and not polymerized into PHF
Summary
Disease Brain abnormal phosphorylation sites have been detected in PHF tau [9, 18,19,20,21,22, 26]. Proteins were eluted to PHF-ubiquitin a t dilutions of 1:50,000 and 1:20,000, respectively; with a 0-1 M NaCl linear gradient in PC buffer, using 10-fold the mAb 2F9 (ascites, Ref. 33) to amyloid Evaluate the contribution of PHF toward the isolated AD P-tau, the Protein concentrations were determined by the modified Lowry 27,000 to 200,000 X g fraction ofAD brain homogenates employed assay of Bensadoun and Weinstein [36]. The resulting pellets were suspended in 100 pl of 10 mM Tris, pH 7.6, and the supernatants dialyzed overnight against the same buffer Both pellets and supernatantswere analyzed by negative stain electron microscopy for the presence of PHF and by slot-blot and immunoblots for ubiquitin and taulevels. Amino acid residues were numbered according to the largest tau isoform, tau 441 [14]
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