Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments.
AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimer's brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimer's brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.
McLaughlin
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Alzheimer's Research Center, Department of Psychiatry, University of Cincinnati College of Medicine, OH 45267, USA.
Zemlan
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FP
Dean
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D000328:Adult; D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology; D000911:Antibodies, Monoclonal; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007536:Isomerism; D009412:Nerve Fibers / Q000378:metabolism*; D016875:tau Proteins / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016875:tau Proteins; D003488:Cyanogen Bromide