Purification and characterization of plasma membrane fractions from cultured pituitary glands.
Highly purified plasma membrane fractions have been prepared from GH3 pituitary cells grown in suspension cultures. These membrane fractions have been obtained by differential and sucrose gradient centrifugation and were characterized in terms of their lipid content, marker enzyme analysis and the binding of 3H-labelled thyrotropin-releasing hormone (TRH) to its receptor. Alkaline phosphatase and 5'-nucleotidase activities were enriched 12-to 15-fold in the plasma membrane fraction with somewhat greater enrichment (28-fold) of the specific binding component for [3H]TRH, with a specific activity of 2286 fmol [3H]TRH bound per mg protein. A single class of binding sites for TRH was observed with an apparent dissociation constant of 18 nM, a value similar to that observed for intact cells. No detectable TRH binding to the nuclear fraction was observed that could not be ascribed to residual plasma membrane contamination. By electron microscopy, these fragments appeared to be sealed vesicles with an average diameter of approximately 1800 A. The binding of 125I-labelled wheat germ agglutinin was used as a marker for plasma membrane purification. In addition to specific binding to this membrane fraction, specific binding was also observed in the nuclear fraction. Studies with fluorescein-labelled wheat germ agglutinin revealed that, in fixed cells, fluorescence was restricted to the plasma membrane. However, if the cells were treated with Triton before labelling, most of the fluorescence was then associated with the cell nucleus. Hence, the use of wheat germ agglutinin binding as a specific membrane marker must be reevaluated.
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D008854:Microscopy, Electron; D009374:Neoplasms, Experimental / Q000648:ultrastructure; D010911:Pituitary Neoplasms / Q000648:ultrastructure*; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis; D011975:Receptors, Mitogen / Q000032:analysis; D018025:Receptors, Thyrotropin-Releasing Hormone
D011956:Receptors, Cell Surface; D011975:Receptors, Mitogen; D018025:Receptors, Thyrotropin-Releasing Hormone; C027503:wheat germ agglutinin receptor