Binding of Radioiodinated Bovine Parathyroid Hormone-(1–84) to Canine Renal Cortical Membranes*

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Abstract
Binding of PTH to purified canine renal cortical membranes was investigated using biologically active radioiodinated bovine PTH-(1–84) [bPTH-(l–84)] as radioligand. PTH-(1–84) is thought to be the major circulating form of bioactive PTH, but oxidative inactivation upon iodination has prevented its use as a radioligand probe of PTH receptors. We have labeled PTH-(l–84) by a microelectrolytic constant current method to a high specific activity (180–220 μCi/μg), corresponding to an average ratio of 1 mol 125/mol peptide. Affinity purification on chick renal membranes consistently improved the radioligandbinding properties, with a 6-fold increase in fraction specifically bound. Analysis of equilibrium (180 min at 15 C) competition curves showed two classes of binding sites for bPTH-(l–84). A high affinity binding site appeared to be coupled to activation of adenylate cyclase and exhibited affinity varying between 1.9 and 4.3 × 108 M-1. The affinity of this site for bPTH was decreased more than 50% by the nonhydrolyzable analog of GTP, guanyl-5′-yl-imidodiphosphate. A low affinity binding site also was detected (Ka = 0.6–6.2 × 106 M-1), and its affinity for bPTH was modulated by the concentration of magnesium. The high affinity sites exhibited hormonal specificity and guanine nucleotide dependency characteristic of peptide hormone receptors, while the low affinity sites did not. Analysis by polyacrylamide gel electrophoresis or high pressure liquid chromatography of the radioligand incubated with or released from purified canine renal cortical membranes revealed a single peak of radioactivity that comigrated with [125I]iodo-bPTH-(1–84). It appears from these studies that cleavage of amino-terminal to residue 43 ([125I]tyrosine) was not required for binding or release of hormone. These experiments indicate that radioiodinated bPTH-(1–84) is a useful probe for further characterization of PTH receptors in kidney and other organs.