IDENTIFICATION AND CHARACTERIZATION OF A TUBULIN BINDING-PROTEIN IN RAT-BRAIN PLASMA-MEMBRANE

Studies on the interaction of FITC-tubulin and I-125-tubulin with isol ated plasma membrane of neural cells and with primary cultures of neur onal (N) and glial (G) cells of rat brain demonstrate the presence of specific, saturable, high affinity tubulin binding sites in these cell s. The positive fluorescence of live unfixed primary cultures of N and G cells following incubation with FITC-tubulin indicate that the tubu lin 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 I-125-tubulin is competed only by tubulin and not by other proteins. Scatchard analysis of the binding of I-125- tubulin to purified plasma membrane indicates very high affinity (K(d) = 85 nM) with a B(max) of 7.4 pmol/mg protein. The putative tubulin r eceptor was partially purified by affinity chromatography on tubulin-s epharose column. Immunoprecipitation of the solubilized tubulin-recept or complex followed by SDS-PAGE analysis and autoradiography, revealed the presence of two components of molecular weights 70 and 45 kDa res pectively, presumably representing the two nonidentical subunits of th e putative receptor. In conjunction with several recent reports indica ting the secretion of high molecular weight proteins from cultured neu ral cells and the ability of tubulin to modulate adenyl cyclase in syn aptic membranes these findings suggest that the binding of exogenous t ubulin to sites external to the plasma membrane may be involved in sig nal transduction.