Covalent labeling of vasopressin receptors from LLC-PK1 cells by the use of a bifunctional reagent

The possibility of covalently attaching vasopressin to its receptors by the use of a bifunctional reagent was explored. Plasma membranes from the LLC-PK1 pig kidney cell line were purified by Percoll density gradient centrifugation. These membranes contained a single population of high affinity (Kd = 5.2 nM) and high capacity (Bmax 3.8 pmol/mg of protein) 3H]lysine vasopressin (3H]LVP)-binding sites. 3H]LVP-labeled receptors could be solubilized with a high yield (83%) and minimal dissociation (9%) by treatment with the non-ionic detergent, octaethylene glycol mono-n-dodecyl ether (C12E8) (0.5%, v/v) in the presence of glycerol (20%). The solubilized 3H]LVP-labeled receptors were stable upon storage at 4 degrees (5% dissociation after 24 hr). They were partially purified to a specific activity of 17 pmol/mg of protein by chromatography on a Cibacron blue-Sepharose column with a yield of 90%. The 3H]LVP-receptor complexes in both intact membranes and the partially purified preparation were almost completely dissociated by incubation at 30 degrees for 30 min in the presence of 20 mM ethylenediaminetetraacetate (EDTA). This property was used to test the effect of ethylene glycol bis (succinimidyl-succinate) (EGS) as cross-linking reagent for the covalent attachment of 3H]LVP to its receptors. After treatment of 3H]LVP-labeled membranes for 30 min with 1 mM EGS at 4 degrees, about 30% of specifically bound 3H]LVP was resistant to EDTA dissociation. The amount of EDTA-resistant binding varied as a linear function of the fractional receptor occupancy and maximal binding capacity of the different batches of membranes used. Similar results were obtained with solubilized and partially purified vasopressin receptors. Upon steric exclusion high performance liquid chromatography, the EDTA-resistant 3H]LVP-labeled material, like the native 3H]LVP-labeled receptor, was eluted as a single and apparently homogeneous peak. The covalent character of the EGS-induced 3H]LVP binding to solubilized or partially purified receptors was assessed by its resistance to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The yield of EGS-induced labeling deduced from these experiments (27%) was close to that determined by the EDTA method. SDS-PAGE analysis of the 3H]LVP-labeled cross-linked material revealed the specific labeling of a major 50-kDa component and a minor component of 30 kDa. The size of these two components was not affected by dithiothreitol.