Solubilization of pituitary receptors for thyrotropin-releasing hormone.
Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent:protein ratios from 0.5 to 20 led to a progressive loss of hormone . receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone . receptor complex was not retained by 0.22 micron filters and remained soluble after ultracentrifugation. Following incubation with high (2.5--10%) concentrations of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent:protein ratio of 0.033. The hormone . receptor complex was included in Sepharose 6B and exhibited an apparent Stoke radius of 46 A in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0 degrees C, while the membrane hormone . receptor complex was stable for up to 5 at 0 degrees C.
D002462:Cell Membrane; D002850:Chromatography, Gel; D003902:Detergents; D010903:Pituitary Gland, Anterior / Q000166:cytology*; D010911:Pituitary Neoplasms; D011092:Polyethylene Glycols; D000644:Quaternary Ammonium Compounds; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D003902:Detergents; D000644:Quaternary Ammonium Compounds; D011956:Receptors, Cell Surface; D011092:Polyethylene Glycols; D013973:Thyrotropin-Releasing Hormone