Atrial natriuretic factor R sub 1 receptor from bovine adrenal zona glomerulosa: Purification, characterization, and modulation by amiloride

Journal Article · Tue Oct 18 04:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00421a025· OSTI ID:6941027

Meloche, S; Ong, H; De Lean, A ^[1]; McNicoll, M; Liu, B ^[2]

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1. Clinical Research Institute of Montreal, Quebec (Canada) Univ. of Montreal, Quebec (Canada)
2. Clinical Research Institute of Montreal, Quebec (Canada)

The atrial natriuretic factor (ANF) R{sub 1} 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 M{sub r} 130,000. Affinity cross-linking of {sup 125}I-ANF to the purified receptor resulted in the labeling of the M{sub r} 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 M{sub r} 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, the authors obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R{sub 1} pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous M{sub r} 130,000 receptor protein displayed high guanylate cyclase activity (1.4 {mu}mol of cyclic GMP formed min{sup {minus}1} (mg of protein){sup {minus}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-R{sub 1} receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.

• OSTI ID: 6941027
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Journal Issue: 21 Vol. 27:21; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADRENAL GLANDS
ANIMALS
BETA DECAY RADIOISOTOPES
BIOCHEMISTRY
BODY
CATTLE
CHEMISTRY
CYCLASES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENDOCRINE GLANDS
ENZYME ACTIVITY
ENZYMES
GLANDS
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LYASES
MAMMALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HORMONES
PROTEINS
PURIFICATION
RADIOISOTOPES
RADIORECEPTOR ASSAY
RECEPTORS
RUMINANTS
TRACER TECHNIQUES
VERTEBRATES