Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride.
The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.
Meloche
S
S
Laboratory of Molecular Pharmacology, Clinical Research Institute of Montreal, Canada.
McNicoll
N
N
Liu
B
B
Ong
H
H
De Léan
A
A
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007700:Kinetics; D008970:Molecular Weight; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D015384:Zona Glomerulosa / Q000378:metabolism*
D011956:Receptors, Cell Surface; D000584:Amiloride; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor