CHARACTERIZATION AND PURIFICATION OF THE BOVINE ADRENAL ANGIOTENSIN-IV RECEPTOR (AT(4)) USING [I-125] BENZOYLPHENYLALANINE-ANGIOTENSIN-IV AS A SPECIFIC PHOTOLABEL

The Ang IV receptor, AT(4), has been shown to play important roles in various mammalian tissues. In this study, structural properties of the AT(4) receptor from bovine adrenals are described using a novel photo active analog of Ang IV, [I-125]Benzoylphenylalanine-Ang IV (BP-Ang IV ), recently developed in our laboratory. [I-125]BP-Ang IV is identical to Ang IV with regards to binding specificity and affinity and is eas ily crosslinked to the AT(4) receptor under UV light, thus greatly fac ilitating the structural analysis of the AT(4) receptor by SDS-PAGE. C omparisons between the native, reduced and nonreduced forms of the AT( 4) receptors by SDS-PAGE revealed that this receptor consists of multi ple subunits. The subunit containing the Ang IV binding site (designat ed as the alpha subunit) has a molecular weight of similar to 165 kDa and contained similar to 20% N-linked carbohydrates. A subunit similar to the adrenal alpha subunit of the AT(4) receptor was identified in all of the bovine tissues examined. Hippocampus and aorta contained ad ditional [I-125]BP-Ang IV bound protein bands with molecular weights o f 150 and 125 kDa, respectively. Further, the alpha subunit was purifi ed to homogeneity using a method that integrates electrofractionation with conventional protein purification techniques.