Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

Abstract

Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclas¢ was restored. Adenylate ¢yclas¢ was stimulated by hCG, hCG + GTP or GppNHp and NaF. Reconstituted proteoliposomcs bound more 12SI-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/ mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG.

Thermal destabilization of ovarian LH/hCG receptors by negatively charged lipids

“…This assumption is supported by studies of reconstitution of the delipidated rat ovarian LH/hCG receptor into proteoliposomes with phospholipids of different charge. Previously we have demonstrated the reconstitution of LH/hCG receptors and adenylylcyclase system from rat ovary (Kolena, 1989). The functional interaction between the receptor and the effector was restored.…”

“…The same buffer was used for elution. After 5-fold dilution the turbid fraction containing proteoliposomes was centrifuged at 160,000g for 60 min (Kolena, 1989;Kolena et al, 1992).…”

Early hCG‐induced desensitization of rat ovarian LH/hCG receptors is associated with altered physical state of membranes

“…Plasma membranes were prepared on the sucrose gradient [8,13]. Reconstitution of the ovarian LH/hCG receptor into proteoliposomes after removal of sodium cholate detergent by absorption on Bin-Beads SM-2 was described previously [13,14]. Isolated luteal cells were prepared by enzymatic dispersion of luteinized ovaries with collagenase [15].…”

“…Adenylylcyclase activity was assayed at 30°C for 25 min with 30~0 gg plasma membrane protein [14,17]. RIA and protein binding methods were used for the determination of progesterone and cAMP concentration [8,9].…”

ProLIF: a quantitative assay for investigating integrin cytoplasmic protein interactions and synergistic membrane effects on proteoliposomes

“…Although bare liposomes can also be produced using extrusion, giving control over the size of the resulting small unilamellar vesicles (SUVs) (Temmerman and Nickel, 2009), this technique does not allow for incorporation of transmembrane proteins. In contrast, detergent removal by Bio-Beads TM is a robust method that has been used to reconstitute many functional transmembrane proteins (Mouro-Chanteloup et al, 2010;Young et al, 1997;Richard et al, 1990;Moriyama et al, 1984;Neves et al, 2009;Lacapère et al, 2001;Geertsma et al, 2008;Kolena, 1989;Nesper et al, 2008;Smith and Morrissey, 2004) resulting in unilamellar vesicles (Rigaud et al, 1995). Such vesicles are close to the detection limit of the scatter of laser light in FACS instruments (Temmerman and Nickel, 2009).…”