Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside.
Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.
Patthi
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S
Salk Institute Biotechnology/Industrial Associates, Inc., La Jolla, California 92037.
Akong
M
M
Veliçelebi
G
G
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D005960:Glucosides; D008168:Lung / Q000378:metabolism*; D008433:Mathematics; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D012995:Solubility
D005960:Glucosides; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D017830:Octoxynol