Dithiothreitol, sodium chloride, and ethylenediaminetetraacetic acid increase the binding affinity of [125I]angiotensin IV to AT4 receptors in bovine adrenal cortex.
The present studies demonstrate that the sulfhydryl reducing agent, dithiothreitol (DTT), increases the specific binding of [125I]angiotensin IV ([125I]AIV) to AT4 receptors in bovine adrenal cortical membranes. Both the degree of stimulation and the pharmacological selectivity of [125I]AIV binding in the presence of DTT were quantitatively different depending on the contents of the assay buffer. Similar effects were also observed using a different sulfhydryl reducing agent, 2-mercaptoethanol (2-MCE). These sulfhydryl reducing agents (100 mM) produced a 200% increase in specific [125I]AIV binding in an assay buffer that has been used to characterize the novel AT4 receptor subtype. A much larger stimulation (700%) of specific [125I]AIV binding was found when the assay was conducted in a buffer that has been used to characterize ligand binding to the AT1 receptor. Ligand association studies indicated that 0.3 nM [125I]AIV displayed similar equilibrium kinetics and stability in both the AT4 and AT1 buffers. Ligand saturation studies indicated that [125I]AIV bound with high affinity (Kd = 6 nM) in the AT4 buffer system, but bound with lower affinity (Kd = 32 nM) in the AT1 buffer system. Removal of NaCl and EDTA from the AT4 buffer also resulted in low-affinity [125I]AIV binding (Kd = 33 nM). The subsequent inclusion of NaCl, EDTA, or DTT resulted in higher-affinity [125I]AIV binding (KdS = 3-14 nM). No significant effects on the apparent density (Bmax) of AT4 receptors were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Jarvis
M F
MF
Rhône-Poulenc Rorer Central Research, Collegeville, PA 19426.
Gessner
G W
GW
D000302:Adrenal Cortex / Q000378:metabolism*; D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D011945:Receptors, Angiotensin / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013438:Sulfhydryl Compounds
C090830:AT4 receptor; D002413:Cations, Divalent; D002414:Cations, Monovalent; D007457:Iodine Radioisotopes; D011945:Receptors, Angiotensin; D013438:Sulfhydryl Compounds; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D012965:Sodium Chloride; D004492:Edetic Acid; D004229:Dithiothreitol