VIP receptors from porcine liver: high yield solubilization in a GTP-insensitive form.
Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.
Voisin
T
T
Unité de recherche sur la différenciation & la Neuroendocrinologie de Cellules Digestives, INSERM U-178, Villejuif, France.
Couvineau
A
A
Guijarro
L
L
Laburthe
M
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002793:Cholic Acids; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure*; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013379:Substrate Specificity; D013388:Succinimides / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D002793:Cholic Acids; D007457:Iodine Radioisotopes; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate