Identification and characterization of a tubulin binding protein in rat brain plasma membrane.
Studies on the interaction of FITC-tubulin and 125I-tubulin with isolated plasma membrane of neural cells and with primary cultures of neuronal (N) and glial (G) cells of rat brain demonstrate the presence of specific, saturable, high affinity tubulin binding sites in these cells. The positive fluorescence of live unfixed primary cultures of N and G cells following incubation with FITC-tubulin indicate that the tubulin binding sites are located on the outer side of the plasma membrane. Such fluorescence was not observed with FITC-BSA, FITC-conalbumin or freshly dissociated cells from rat tissues or established cell lines. Binding of FITC-tubulin or 125I-tubulin is competed only by tubulin and not by other proteins. Scatchard analysis of the binding of 125I-tubulin to purified plasma membrane indicates very high affinity (Kd = 85 nM) with a Bmax of 7.4 pmol/mg protein. The putative tubulin receptor was partially purified by affinity chromatography on tubulin-sepharose column. Immunoprecipitation of the solubilized tubulin-receptor complex followed by SDS-PAGE analysis and autoradiography, revealed the presence of two components of molecular weights 70 and 45 kDa respectively, presumably representing the two nonidentical subunits of the putative receptor. In conjunction with several recent reports indicating the secretion of high molecular weight proteins from cultured neural cells and the ability of tubulin to modulate adenyl cyclase in synaptic membranes these findings suggest that the binding of exogenous tubulin to sites external to the plasma membrane may be involved in signal transduction.
Department of Cell Biology, Indian Institute of Chemical Biology, Calcutta.
S
S
Chaudhury
S
S
Das
S K
SK
Banerjee
P K
PK
Sarkar
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D012995:Solubility; D014404:Tubulin / Q000378:metabolism
D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D014404:Tubulin; C088298:tubulin receptor, rat; D011092:Polyethylene Glycols; D017830:Octoxynol; D016650:Fluorescein-5-isothiocyanate