Purification and properties of active atrial-natriuretic-peptide receptor (type C) from bovine lung.
Atrial-natriuretic-peptide (ANP) receptor, previously identified as a 140 kDa protein with a disulphide-linked homodimeric structure, was purified from bovine lung by (NH4)2SO4 fractionation and affinity chromatography on ANP-Affi-Gel 10. The purified receptor had a binding capacity of 4.2 nmol of ANP/mg of protein and an affinity constant of 6.5 pM. The isoelectric point of the receptor was 5.8, consistent with the acidic nature of the protein (amino acid analysis revealed a predominance of glutamic acid and aspartic acid residues). Treatment with endoglycosidase H and glycopeptidase F revealed that the receptor has three complex types of oligosaccharide chains per 70 kDa subunit. Deglycosylation of the receptor did not affect its binding activity. Reduction with dithiothreitol and reoxidation by dialysis revealed a strong tendency of the receptor subunits to dimerize via disulphide cross-linking; however, carboxymethylation of the reduced receptor indicated that the intersubunit disulphide bond is not necessary for the ligand-binding activity.
Uchida
K
K
Department of Biological Sciences, Tokyo Institute of Technology, Japan.
Mizuno
T
T
Shimonaka
M
M
Sugiura
N
N
Nara
K
K
Ling
N
N
Hagiwara
H
H
Hirose
S
S
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides; D006023:Glycoproteins / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007526:Isoelectric Point; D008024:Ligands; D008168:Lung / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D013329:Structure-Activity Relationship
D004220:Disulfides; D006023:Glycoproteins; D007477:Ions; D008024:Ligands; D011956:Receptors, Cell Surface; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor