Abstract
Tau (tau) is a major constituent of paired helical filaments (PHF) found in Alzheimer's disease. The current study examines the possibility that the distinct properties of PHF-associated tau proteins (tau PHF) result from post-translational modifications of normal soluble tau (tau s). Following hydrofluoric acid (HF) treatment, tau PHF proteins are heat- and acid-stable, soluble in 2-(N-morpholino)ethanesulfonic acid buffers and display the same molecular weight, pI, and immunochemical properties as normal tau s. Alkaline phosphatase treatment of dissociated PHF results in similar, although less extensive, electrophoretic changes and a reduction in PHF-1 immunoreactivity. Therefore, phosphorylation of normal tau s appears to be responsible for the distinct properties of tau PHF. Although our results suggest that all of the normal tau isoforms are in PHF, the relative abundance of individual tau species differs in HF-treated PHF and tau s samples. Moreover, the loss of PHF following HF treatment suggests that post-translational modifications contribute to the structural stability of PHF.
Highlights
Individual TPHF proteins are more clearly resolved by equilibrium isoelectric focusing (6), the basic T@ proteins are more consistently resolved by nonequilibrium isoelectric focusing (4)
We used two-dimensional nonequilibrium pH gradient gel electrophoresis (19) to analyze T. and TPHF followingdifferent dephosphorylation conditions. 5-20 pg of methanol-precipitated proteins were resuspended in 8 M urea, 0.5% SDS k 1% 8-mercaptoethanol and mixed with an equal volume of lysis buffer consisting of 8 M urea, 8% Triton X-100, and 2% ampholines (Pharmacia LKB Biotechnology Inc.)
The PI of T was determined by using the pHgradient from a standard nonequilibrium pH gel and phosphocellulose-purifiedbovine tubulin as an Dephosphorylation of TpHF-Fig. 1 is an Alz 50-reactive immunoblot of PHF which has been dephosphorylated under different conditions
Summary
EGTA, [ethylenebis(oxyethylenenitrilo)tetraacetic acid; PPi, sodium enized with 10 volumes of 50 mM Tris, 1%SDS, 1%8-mercaptoethpyrophosphate; NFT, neurofibrillary tangles; Chaps, 3-[(3-cholami- anol, pH 7.4, incubated a t 37 “C for 2 h, heated at 100 “C for 5 min, dopropyl)dimethylammonio]-1-propanesulfonicacid 7pHF Proteins rpm for 35 min in a Ti-60 rotor (Beckman Instruments, Inc.), PHF in thepellet was further purified by sucrose density centrifugation or by washing the pellet with H buffer containing 1%p-mercaptoethano!, 1%sarkosyl, and/or 0.1 M MES, 1 mM EGTA, 0.1 mM EDTA, 0.5 mM MgCl,, 1 mM 0-mercaptoethanol, pH 6.8 (MES buffer) These procedures resulted in relatively impure preparations with low yields, SDS-insoluble PHF were visible by electron micros-
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