Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver.
Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.
Guijarro
L
L
Unité de Recherches sur la Différenciation et la Neuroendocrinologie des Cellules Digestives, INSERM, Villejuif, France.
Couvineau
A
A
Calvo
J R
JR
Laburthe
M
M
D000818:Animals; D002793:Cholic Acids; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide
D002793:Cholic Acids; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate