Comparison of methods for the in vitro assembly of postmortem human brain microtubules that retain the microtubule-associated protein tau.
Several methods for the in vitro assembly of microtubules from postmortem human brain were compared for the purpose of obtaining microtubule preparations that best retained their microtubule-associated proteins. The polymerized microtubules from the preparations were examined by negative staining and electron microscopy and shown to consist of well-formed microtubules with varying amounts of abnormal assembly products that differed between methods. The microtubule protein was analyzed by SDS-polyacrylamide gel electrophoresis, quantitative densitometry, as well as trans-blotted onto membranes which were reacted with monoclonal antibodies to tubulin subunits and microtubule-associated proteins. All the preparations were found to contain both the alpha- and beta-tubulin subunits with quantitative differences, but they varied most, both quantitatively and qualitatively, in their content of microtubule-associated proteins. The optimal method for the assembly of soluble tubulin from postmortem human brain cytosol into intact microtubules which specifically retained most of their MAPs, especially tau, employed 4 M glycerol assembly buffer in the presence of 10 microM taxol and 1 mM GTP. The isolation methods were used to compare young and aged brains, and there were fewer microtubule-associated proteins, especially tau, associated with the microtubules in advanced age, in all preparations.
Sparkman
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Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235-9072.
D000293:Adolescent; D000368:Aged; D000369:Aged, 80 and over; D001921:Brain / Q000648:ultrastructure*; D001923:Brain Chemistry; D002474:Cell-Free System; D002648:Child; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D014021:Tissue Preservation; D016875:tau Proteins / Q000378:metabolism* / Q000648:ultrastructure
D008869:Microtubule-Associated Proteins; D016875:tau Proteins