Molecular characterization of the solubilized atrial natriuretic factor receptor from bovine adrenal zona glomerulosa.
The atrial natriuretic factor (ANF) receptor has been solubilized from bovine adrenal zona glomerulosa membranes with the nonionic detergent octyl-beta-D-glucoside. Mathematical analysis of competition binding curves with solubilized receptor revealed the presence of two classes of binding sites with pK of 10.4 (Kd = 40 pM) and 8.2 (kd = 6000 pM), similar to the native receptor of intact membranes. The hydrodynamic properties of the ANF receptor were determined by prelabeling the membrane receptor with 125I-ANF prior to solubilization. The solubilized 125I-ANF-receptor complex eluted as a major peak with a Stokes radius of 50.8 A from a Superose 6 steric exclusion column. A partial specific volume of 0.770 ml/g and a sedimentation coefficient (S20,w) of 6.34 S were determined by sucrose density gradient centrifugation in H2O and D2O. These data were used to calculate a molecular weight of 158,000 and a frictional ratio of 1.25 for the labeled receptor-detergent complex. The amount of detergent bound to the receptor was estimated to be 0.45 g/g of protein, assuming a partial specific volume of 0.730 ml/g for the protein. Correction for the mass contributed by the bound detergent yielded a molecular weight of 109,000 for the receptor protein. Affinity cross-linking of 125I-ANF to its binding sites in zona glomerulosa membranes and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that one single band with apparent Mr 130,000 was specifically labeled. These results indicate that the ANF receptor from bovine adrenal cortex is a membrane protein with a total molecular weight of 110,000-130,000 and suggest that the native protein contains only one single polypeptide chain.
D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor