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 (MAP) in pituitary tissue and in a cultured pituitary cell line examined. To isolate MAP from the tissue, previously described taxol-dependent method was modified for purifying microtubules and MAP. 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 endogneous tubulin. Pituitary MAP 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 MAP 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 cells by using a slight modification of the previously described taxol procedure. Immunoblotting indicated once again that MAP 1A, MAP 1B, and MAP 2 were included among the MAP. These results were further confirmed by immunofluorescence microscopy of GH3 cells and sections of rat anterior pituitary tissue. The first system was identified other than brain containing significant levels of several of the high MW MAP characteristic of brain tissue, suggesting that microtubules in neurons and secretory cells may be involved in at least some related functions. Preparations of both secretory tissue and secretory cell line microtubule are available for further in vitro investigation of the interaction of microtubules with secretory granules.