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)