Open AccessPurification and characterization of a new, ubiquitously distributed class of microtubule‐associated protein with molecular mass 250 kDa
Author(s) -
Katsuki Miho,
Tokuraku Kiyotaka,
Nakagawa Hiroyuki,
Kotani Susumu
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01822.x
Subject(s) - microtubule , nocodazole , tubulin , microtubule associated protein , cytoplasm , molecular mass , polyclonal antibodies , western blot , biology , microbiology and biotechnology , cytosol , blot , immunofluorescence , microtubule polymerization , negative stain , cytoskeleton , biochemistry , electron microscope , antibody , cell , gene , genetics , enzyme , physics , optics
A heat‐stable microtubule‐associated protein (MAP) with relative molecular mass 250 000, termed 250‐kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250‐kDa MAP was able to bind to taxol‐stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western‐blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250‐kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250‐kDa MAP colocalized with free tubulin. Under electron microscopy, 250‐kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.