Identification of high molecular weight microtubule-associated proteins in anterior pituitary tissue and cells using taxol-dependent purification combined with microtubule-associated protein specific antibodies.

Abstract

The composition of microtubule-associated proteins (MAPs) in pituitary tissue and in a cultured pituitary cell line was examined. To isolate MAPs from the tissue, we modified our previously described taxol-dependent method for purifying microtubules and MAPs [Vallee, R. B. (1982) J. Cell Biol. 92, 435-442]. Microtubules were assembled from purified calf brain tubulin with the aid of taxol and were then added to a cytosolic extract of pituitary tissue which did not support microtubule assembly from endogenous tubulin. Pituitary MAPs bound to the microtubules and were isolated by centrifugation. Polypeptides corresponding in electrophoretic mobility to the component proteins of MAP 1 were among the major MAPs observed. Immunoblotting of the pituitary MAP fraction revealed the presence of appreciable quantities of MAP 1A, MAP 1B, and MAP 2 polypeptides. Microtubules were also prepared from GH3 rat pituitary tumor cells by using a slight modification of our previously described taxol procedure. Immunoblotting indicated once again that MAP 1A, MAP 1B, and MAP 2 were included among the MAPs. These results were further confirmed by immunofluorescence microscopy of GH3 cells and sections of rat anterior pituitary tissue. Thus, we have identified the first system other than brain containing significant levels of several of the high molecular weight MAPs characteristic of brain tissue, suggesting that microtubules in neurons and secretory cells may be involved in at least some related functions. In addition, we now have available preparations of both secretory tissue and secretory cell line microtubules for further in vitro investigation of the interaction of microtubules with secretory granules.